WO2022027088A1

WO2022027088A1 - Lateral flow device and uses thereof

Info

Publication number: WO2022027088A1
Application number: PCT/AU2021/050839
Authority: WO
Inventors: Dale GODFREY; Damian Purcell; Nicholas GHERARDIN; David Anderson; Tom FULFORD; Huy VAN; Shuning ZHENG
Original assignee: The University Of Melbourne; The Macfarlane Burnet Institute For Medical Research And Public Health Ltd
Priority date: 2020-08-03
Filing date: 2021-08-02
Publication date: 2022-02-10

Abstract

The present disclosure provides a lateral flow assay (LFA) device for detecting neutralising antibodies against SARS-CoV-2 in a biological fluid sample, and uses thereof. The assay uses competition of the neutralising antibodies present in the sample with the ACE2-RBD on the test strip, leading to binding inhibition and decrease in signal intensity in the test region.

Description

LATERAL FLOW DEVICE AND USES THEREOF

FIELD OF THE DISCLOSURE

[0001] The disclosure relates generally to a lateral flow device for detecting the presence of biomarkers of an immune response to viral infection, in particular neutralising anti-viral antibodies.

BACKGROUND

[0002] Immunoassays are useful for detecting the presence of analytes in a sample, and in some instances allow the level of the molecule of interest to be quantified, either as an absolute amount or relative to a reference value, typically employing binding reagents, such as immunoglobulin or antigen-binding fragments thereof, that specifically bind to the analyte of interest. However, traditional immunoassays are generally time consuming and labour intensive, typically requiring advanced laboratory equipment and skilled personnel. Recent technological advances have allowed immunoassays to be sufficiently miniaturized and compartmentalized into, for example, chromatography-based test strips, which can be employed and used by unskilled or non-healthcare workers. This is particularly advantageous for rapid, point-of-care (POC) diagnosis or monitoring of health and disease. However, traditional chromatography-based test strips, which use techniques of chromatography to separate components as a solvent front moves upward through a substrate, do not always provide the requisite selectivity for identifying analytes of interest. [0003] More recently, lateral flow assays (LFA) have been developed, which utilise capillary action to move the solvent front laterally, across the length of a test strip. LFA make use of the force of capillary action to draw a solvent, in a lateral fashion, through capillary beds formed in or on a substrate through a series of active regions on the test strip to provide a complete immunoassay reaction and a recognizable result at a defined region along the test strip.

[0004] LFA is a chromatography-based platform for the detection and quantification of analytes, including in complex mixtures, where the sample is placed on a test device and the results are displayed within minutes, typically from about a few to about 30 minutes (see, e.g., Koczula and Gallotta, 2016, Essays Biochem:, 60(1): 111-120). Low development costs and ease of production mean that LFA can be deployed in multiple fields in which rapid tests are required, such as in hospitals, physician's offices and clinical laboratories for the qualitative and quantitative detection of analytes. LFA have also been used to screen for human and animal diseases, pathogens, chemicals, toxins and water pollutants. Perhaps the most widely spread and recognised application of LFA is the home pregnancy test kit. Moreover, because of the long shelf life and the fact that refrigeration is generally not required for storage, LFA are very well adapted for use in remote regions, developing countries, small ambulatory care settings and battlefields. Because of their portability, ease of use and low cost, LFA can be used to identify and monitor exposure to disease -causing pathogens, such as bacteria, parasites and viruses, including outbreaks in wider communities. However, in this context, most LFA are limited in their application, insofar as they will only provide information on whether a subject has been previously exposed to a pathogen, or a pathogen-derived antigen, and to which an antibody response has been raised. From a clinical perspective, that information can be insufficient because it is not indicative of whether the subject has developed sufficient protective immunity, including against subsequent infection by the pathogen. For instance, currently available antibody tests for SARS-CoV-2 infection measure IgG, IgM, IgA and/or total antibodies to one or more viral proteins. Notwithstanding that these tests are typically associated with high false positive rates (1-3%), true positive results are only indicative of prior infection with the virus and are unable to discern individuals who are likely to have developed at least some protective immunity against the virus, either through prior exposure to the virus or to a viral antigen (e.g., as a vaccine) that is designed to raise a protective antibody response to the virus.

[0005] Hence, whilst there have been significant advances in LFA, there remains an urgent need for improved LFA formats and devices, in particular those that solve, or at least partly alleviate, some of the aforementioned limitations.

SUMMARY

[0006] In an aspect disclosed herein, there is provided a lateral flow assay (LFA) device for detecting an anti-viral antibody in a biological fluid sample, the device comprising, in the direction of flow:

(a) a sample application region, wherein the sample application region is configured to receive a biological fluid sample; (b) a conjugation region comprising a tracer antigen, wherein the tracer antigen comprises a detectable moiety; and

(c) a test region comprising an immobilised capture antigen wherein the device is configured to move the biological fluid sample by capillary action in the direction of flow from the sample application region to the test region; wherein the tracer antigen or the immobilised capture antigen is a viral antigen or a functional variant thereof; and wherein

(i) in the absence of an anti-viral antibody in the biological fluid sample, a complex comprising the tracer antigen and the capture antigen is formed to produce a detectable test signal at the test region; and

(ii) in the presence of an anti-viral antibody in the biological fluid sample, the anti-viral antibody inhibits the formation of the complex comprising the tracer antigen and the capture antigen at the test region, thereby producing a weaker test signal at the test region when compared to a reference signal that is representative of a test signal that is produced in absence of the anti-viral antibody.

[0007] In another aspect disclosed herein, there is provided a method of detecting an antiviral antibody in a biological fluid sample of a subject, the method comprising:

(a) applying a biological fluid sample from a subject to the sample application region of the lateral flow assay device, as described herein, for a period of time sufficient to allow the biological fluid sample and trace antigen to flow by capillary action to the test region; and

(b) comparing the detectable test signal at the test region with a reference test signal; wherein a weaker test signal at the test region when compared to the reference test signal is indicative of the presence of the anti-viral antibody in the biological fluid sample.

[0008] In another aspect disclosed herein, there is provided a method of identifying a subject as being a source of neutralising anti-viral antibodies, the method comprising: a) obtaining a biological fluid sample from a subject; b) applying the biological fluid sample from step (a) to the sample application region of the lateral flow assay device, as described herein, for a period of time sufficient to allow the biological fluid sample and trace antigen to flow by capillary action to the test region; and c) comparing the detectable test signal at the test region with a reference test signal, wherein the subject is identified as a source of neutralising anti-viral antibodies when a weaker test signal is detected at the test line when compared to the reference test signal.

[0009] The present disclosure also extends to a composition enriched for neutralising antiviral antibodies obtained from the source identified by the method disclosed herein.

[0010] In another aspect disclosed herein, there is provided a method of treating or preventing viral infection in a subject in need thereof, the method comprising administering to the subject the composition described herein.

[0011] The present disclosure also extends to a method of identifying neutralising anti-viral antibodies in a sample, the method comprising: a) applying a sample to the sample application region of the lateral flow assay device, as described herein, for a period of time sufficient to allow the sample and trace antigen to flow to the test region; and b) comparing the detectable test signal at the test region with a reference test signal, wherein the subject is identified as a source of neutralising anti-viral antibodies when a weaker test signal is detected at the test region when compared to the reference test signal.

BRIEF DESCRIPTION OF THE FIGURES

[0012] FIGURE 1 shows detection of immobilised SARS-CoV-2 Receptor Binding Domain (RBD) and titration of the amount of RBD fused to IgG Fc domain (RBD-Fc) or conjugated to biotin (RBD-biotin) that was required to allow visual detection using anti-IgG or antibiotin antibodies. Equal volumes of three different concentrations of RBD-Fc / RBD-biotin (0.01, 0.1 and 1 mg/mL) were spotted onto the strip, and detected using gold-conjugated anti-IgG or anti-biotin antibodies. The binding of gold conjugated antibodies was indicated by the red colour. This showed that RBD-Fc or RBD-biotin can be detected in a lateral flow format when spotted at a concentration of Img/ml, and sub-optimally at 0. Img/ml.

[0013] FIGURE 2 shows titration of the amount of biotin labelled Angiotensin Converting Enzyme 2 (ACE2 -biotin) required to detect Ipg of immobilised RBD-Fc. Ipg of RBD-Fc was immobilised onto the strip and exposed to three different concentrations of ACE2 -biotin (25, 50 and 100 pg/mL). A control spot, which was not exposed to any ACE2 -biotin, was included (bottom strip). Bound ACE2 -biotin was detected using gold-conjugated anti-biotin antibody. The control strip was incubated with gold-conjugated anti-IgG to detect the presence of RBD-Fc. This indicated that ACE2 -biotin could bind to immobilised RBD giving a similar signal for each of the concentrations tested down to 25pg/ml.

[0014] FIGURE 3 shows a further titration experiment to determine minimum of the amount of biotin labelled ACE2 (ACE2 -biotin) required to detect 1 pg of immobilised RBD-Fc. 1 pg of RBD-Fc was immobilised onto the strip and exposed to five different concentrations of ACE2-biotin (50, 5, 0.5, 0.05 0.005 pg/mL). Bound ACE2 -biotin was detected using gold- conjugated anti-biotin antibody. This indicated that ACE2-biotin could bind to immobilised RBD giving a clear signal at 50pg/ml but a diminished signal at 5pg/ml. This suggests that the optimal concentration of ACE2 -biotin was between 5 and 25pg/ml.

[0015] FIGURE 4 shows healthy human (no known infection with SARS-CoV-2) serum blocking of ACE2-RBD binding. Ipg of RBD-Fc was immobilised onto the strip. These spots were then exposed to ACE2 -biotin that was pre-incubated with different dilutions of healthy human serum. Binding of ACE2-biotin to immobilised RBD-Fc was determined using gold-conjugated anti-biotin antibody. Only at the highest concentration (80% sera dilution) of the healthy human serum, was there visible inhibition of the gold-conjugated anti-biotin antibody signal (arrow). This is expected because at very high concentrations of human sera approaching undiluted sera, non-specific inhibition of molecular interactions may occur. This suggested that healthy human serum could be used at concentrations below 80% in order to avoid non-specific inhibition of binding of ACE2-biotin to immobilised RBD-Fc. It was subsequently determined that if serum was run first, and then ACE2 -biotin was run, there was no inhibitory effect due to high concentrations of serum up to at least 40% (see FIGURE 7).

[0016] FIGURE 5 shows that a serum sample from a COVID- 19 patient inhibited binding of ACE2 to RBD. Having established the basic conditions that allowed detection of ACE-2 bound to RBD-Fc, strips were made with a dedicated striping machine. RBD-Fc striped (lug/ml) strips were incubated with a 20% dilution of serum samples from a healthy human (206) or from a COVID-19 patient (302) before being exposed to ACE2 -biotin (0.5pg/ml). Level of ACE2-biotin binding to immobilised RBD-Fc was determined using gold- conjugated anti-biotin antibody. Some reduction in the signal could be observed, indicating that the COVID-19 patient sample 302 comprises anti-viral antibodies that could bind to the immobilised RBD and inhibit the binding of ACE2 -biotin to immobilised RBD-Fc. This result shows that a serum sample from a COVID- 19 patient inhibited binding of ACE2 to RBD, but the reduction in signal was not very strong.

[0017] FIGURE 6 shows a clear titration of ACE2 -biotin was detectable down to 0.5pg/ml. Because the reduction in signal was not very dramatic, it was reasoned there may be too much RBD striped on the strip. Therefore, three different amounts of RBD-Fc (1.0, 0.3 and O. lug/ml) striped onto strips were tested. These strips were tested at three different concentrations of ACE2 -biotin (5.0, 0.5, 0.05pug/ml). These results showed a clear titration of ACE2 -biotin that was detectable down to 0.5pg/ml, for each of the RBD concentrations. However, the signal grew fainter as the RBD concentration decreased. Because we did not want a signal that was too strong, and more difficult to inhibit, these data suggested that 0.3pg/ml RBD striped strips was ideal and 0.5pg/ml ACE2 was ideal.

[0018] FIGURE 7. In order to increase the amount of antibody in the samples tested, a higher percentage of serum was tested. Using the conditions established in Figure 6, a range of healthy human serum concentrations (0, 10, 20 and 40%) were tested to determine the highest amount that could be use before non-specific inhibition of ACE2-RBD binding was observed. Two approaches were used: (i) where the human serum was pre-run on the strips, followed by ACE2 -biotin mixed with anti-biotin-gold; and (ii) where the human serum was mixed with ACE2 -biotin and anti-biotin-gold and they all were run together. These data showed that up to 40% serum did not interfere with the ACE2-RBD interaction providing the serum was pre-run. If the serum was mixed with ACE2, then only 10% serum could be tolerated.

[0019] FIGURE 8. Using the new conditions established, the ability of 40% serum from a COVID-19 patient to inhibit RBD-ACE2 binding was tested. Three different formats were tested: 1. 0.3pg/ml RBD-Fc stripe, ran 40% serum, detected w 0.5pg/ml ACE2; 2. O. lpg/ml RBD-Fc stripe, ran 40% serum, detected w 0.5pg/ml ACE2; 3. 0.3pg/ml RBD-Fc stripe, run 10% serum + ACE2 -biotin + anti -biotin-gold. 40% COVID- 19 patient serum seemed to cause a stronger reduction in the signal compared to 10% serum, suggesting that the amount of antibody in serum is limiting.

[0020] FIGURE 9 shows that COVID- 19 sera inhibit binding of ACE2 to RBD. Strips with immobilised RBD-Fc were incubated with 60% dilution of serum samples from healthy human or COVID-19 patient before being exposed to ACE2 -biotin. Some reduction in the signal could be observed with the serum samples from the COVID-19 patients compared to the samples from healthy donors. These samples were read on an Axxin strip reader instrument giving numerical values for stripe intensity. These data are also graphed as % inhibition based on inhibition of the mean signal intensity from the healthy control samples. This result confirms that COVID- 19 patient samples comprise anti-viral antibodies that can inhibit the binding of ACE2-biotin to immobilised RBD-Fc, suggesting the presence of neutralising antibodies.

[0021] FIGURE 10 shows that anti-RBD antibodies in COVID-19 patient serum can be directly detected using the LFA strips described herein. Strips with immobilised RBD-Fc were exposed to 10% dilution of COVID- 19 patient serum. A control strip was exposed to no serum. In the absence of healthy serum, it was observed that the gold-conjugated antihuman IgG binds to the RBD-Fc directly. This was not observed in the presence of serum. Thus, antibodies to RBD of SARS-CoV-2 in COVID-19 patient serum, but not healthy serum samples, could be detected using gold conjugated goat-anti-human IgG.

[0022] FIGURE 11 shows immobilisation of ACE2-biotin in an attempt to invert the interaction so that ACE2 is bound to the strip and RBD flows over the ACE2 with the sample components. Equal volumes of three different concentrations of ACE2 -biotin (0.028, 0.28 and 2.8 mg/mL) were spotted onto the strip. This result showed that ACE2-biotin does not immobilise very effectively to the strip and appears to diffuse away.

[0023] FIGURE 12 tests for detection of ACE2 with RBD. 1 pg of ACE2 -biotin or ACE2 without a biotin tag, was spotted onto each strip, and exposed to different concentrations of RBD-Fc. A control experiment using a test strip with ACE2 biotin was incubated with blank running buffer. The interaction was assayed using gold conjugated anti -IgG for the test strips or gold conjugated anti-biotin for the control strip was used to detect for the binding of RBD- Fc to strip. No binding was observed, indicating that no RBD-Fc was bound. The control strip had a signal, showing anti-biotin antibody interacted with ACE2-biotin suggesting that ACE2-biotin had bound. This suggested that RBD-Fc could not bind to strip-bound ACE2- biotin, suggesting that strip-bound ACE2 -biotin had somehow changed its conformation.

[0024] FIGURE 13 shows immobilisation of ACE2-biotin to the strip using streptavidin. This approach was tried as an alternative way to immobilise ACE2, since the previous experiments suggested that it did not retain the ability to bind to RBD when it was directly immobilised to the strip. (A) Equal volumes of streptavidin (SAV) or streptavidin- Phycoerythrin (SAV-PE) of different concentrations were spotted onto the strip and incubated with biotin. The streptavidin-biotin complex was then detected using gold- conjugated anti-biotin antibodies. Ipg streptavidin could be detected. (B) Ipg streptavidin was spotted onto the strip and incubated with ACE2 biotin (50pg/mL). Strips were then exposed to three different amounts of RBD (0, 0.5, 5 and 50 pg) and detected using gold- conjugated anti-IgG antibodies. RBD binding could be detected at each concentration but 5ug appeared to be optimal, whereas 0.5 was very faint.

[0025] FIGURE 14 shows that COVID- 19 sera samples inhibit binding of RBD-Fc to streptavidin immobilised ACE2-biotin. ACE2-biotin was immobilised on the strip using streptavidin. (A) Strips were then run with two human COVID- 19 sera samples (322-B and 325-B) or two healthy control sera samples (039, 046), each diluted at 60% and run on the strip mixed with 5pg/ml RBD-Fc. A no sera control was also included. While the intensity of the signals were very faint, even in the absence of sera or in presence of sera from healthy controls, inhibition was observed with the COVID- 19 sera samples. (B) With encouragement from this experiment, we had some strips striped up with streptavidin so we could quantify the signal on the Axxin strip reader. The experiment was repeated in a similar manner to A, using streptavidin striped strips, and a panel of 11 healthy human sera samples and a panel of 10 COVID-19 patient sera. It was found that the signals even from the control samples were very weak and diffused/faded very quickly, suggesting the ACE2 was not well-bound to the strips. This made it very difficult to ascertain whether inhibition of signal was being detected in the presence of COVID-19 patient sera.

[0026] FIGURE 15 shows that binding between immobilised ACE2-biotin and RBD-Fc can be inhibited with soluble ACE2. Reverted to the spotting technique for binding ACE2 -biotin to streptavidin spotted strips, streptavidin immobilised ACE2 -biotin was exposed to RBD- Fc pre-incubated with soluble, non-biotinylated human ACE2 at different molar ratios. The results showed that excess soluble human ACE2 prevented RBD-Fc binding to immobilised ACE2-biotin.

[0027] FIGURE 16 shows that Fc-ACE2 adheres to the strip efficiently, and allows dose- responsive visualisation of RBD binding. A different approach was taken to bind ACE2 to the strip, using Fc-ACE2. (A) Different amounts (4, 2, 1, 0.5 0.25 and 0 pg) of ACE2 fused to Fc were spotted onto the test strip and detected using gold-conjugated anti-human IgG. The result showed that Fc-ACE2 adheres to the strip very well. (B) 2pg of Fc-ACE2 is spotted on to the strip and then exposed to three different concentrations of biotinylated RBD (RBD-biotin). Detection of bound RBD-biotin was visualised using gold-conjugated antibiotin antibody. The signal showed a clear dose-response change in intensity, which correlated with the titration of RBD-biotin. (C) The experiment was repeated using spots of either 2pg or 0.26pg of Fc-ACE2, which were then exposed to two concentrations of RBD- biotin (2.4pg/mL or 24pg/mL). A clear signal was seen for the 2pg of Fc-ACE2 spot while a very faint signal could be seen for the 0.26pg of Fc-ACE2 spot. In each case they did not show a dose-response to RBD-biotin suggesting that the lower dose of 2.4pg/ml RBD biotin was saturating.

[0028] FIGURE 17 shows the titration of healthy patient sera to determine if high concentrations of healthy sera could non-specifically interfere with the Fc-ACE2-RBD interaction. 1 pg of Fc-ACE2 was spotted on to the test strip and then exposed to RBD-biotin that was pre-incubated with healthy human sera of increasing concentrations. No signal inhibition could be seen, even at 80% serum dilution. A serum sample with no RBD control experiment gave no signal.

[0029] FIGURE 18 shows that COVID-19 patient sera could inhibit the interaction between immobilised Fc-ACE2 and RBD-biotin. 1 pg of Fc-ACE2 is spotted on to the strip and then exposed to RBD-biotin that was pre-incubated with healthy human sera or COVID- 19 sera that was independently confirmed to contain neutralising antibodies. Detection of RBD- biotin bound to immobilised Fc-ACE2 using gold-conjugated anti-biotin antibodies indicated that the COVID-19 sera strongly inhibited signal formation.

[0030] FIGURE 19 shows the titration of RBD-biotin to ensure that the presence of any biotin (vitamin B7) in the serum of COVID- 19 patients does not affect the test. RBD-biotin was saturated with anti-biotin-Au. Signal intensity was measured (pre-sera). Then 30pL of healthy uninfected human sera or COVID-19 patient sera was then flowed onto the spot, and washed with 60 pL of wash buffer (A). Signal intensity was measured (post-sera). No significant reduction in signal intensity could be detected pre- and post-sera measurements (using Image J software to graph data as in (B)).

[0031] FIGURE 20 shows the titration of RBD-biotin amounts to optimise signal to noise ratio. Fc-ACE2 was immobilised as stripes on the strip, at different concentrations (2mg/mL, and 0.8mg/mL). 4 different concentrations of RBD-biotin was flowed across the Fc-ACE2 lines, before incubation with 30uL anti-biotin-Au for 10 minutes (Figure 17 A). Quantification of the signals is depicted in the graph of Figure 17B. [0032] FIGURE 21 shows that immobilised Fc-ACE2 and RBD-biotin can discern between sera with and without neutralising anti-SARS-CoV-2 antibodies, and also for antibodies from different species. The strips with immobilised Fc-ACE2 were tested with 5 serum samples: 1) Healthy uninfected human serum sample, 2) COVID- 19 patient serum sample, 3) known negative mouse serum sample (healthy mouse (1.5)), 4) RBD-immunised-positive mouse serum sample (immunised mouse (4.2)) and 5) pre-bleed sample of immunised mouse (that is sample taken before immunisation). The 0.8mg/mL ACE2 band gave only a faint signal. However, the 2mg/mL showed visible signal reduction with the COVID-19 patient sample, and similar reduction was seen with the RBD-immunised mouse serum sample. The signal intensity is shown in Figure 2 IB.

[0033] FIGURE 22 shows RBD-immunised mouse serum sample (mouse 4.2) and a control non-RBD-immunised mouse serum sample (mouse 1.5) used to titrate the amount of RBD- biotin for this assay. Different dilutions of COVID- 19-positive sera were also compared. Only the COVID- 19-positive or RBD-immunised mouse serum samples gave visible reduction in signal, and this was true of both 20% and 5% dilutions of the serum sample.

[0034] FIGURE 23 shows a blinded panel of human sera tested using the lateral flow assay established and tested as shown in FIGURE 22, for anti-SARS-CoV-2 neutralising antibodies. Quantification for all samples were performed blind so we were unaware of which samples contained anti-SARS-CoV-2 antibodies. After performing the assay, the decoded samples revealed that all samples were from CO VID-19 patients, but two of these 72 samples had undetectable/background levels of RBD specific IgG antibodies (independently tested). These two negative samples are boxed (in red). The signal intensity data from the strip tests in (A) were read on the Axxin strip reader, quantified and peak intensity values for 2mg/mL Fc-ACE2 immobilised test regions in order from highest to lowest intensity (B) The two negative samples are encircled and their anti-RBD antibody titres shown in red (independently determined). Anti-RBD antibody titres (independently determined) for the most inhibited samples are also shown (B). After the experiment was unblended, and it was found that there were only two negative samples, an additional experiment was performed with additional samples including 10 healthy control serum samples, 8 additional COVID- 19+ samples, plus 4 samples from the experiment shown in 23A in order to normalise between the two experiments. These data were used to generate a graph showing the % of RBD-ACE2 -binding inhibition in COVID- 19-positive and -negative samples where 0% is determined by the mean signal intensity of the healthy control samples. An additional experiment was performed with additional samples including 10 healthy control serum samples, 8 additional COVID- 19+ samples, plus 4 samples in order to normalise between the two experiments (Fig 23C). The collective data derived from the experiment in Figures 23A and 23C were normalised and used to generate a graph showing the % of RBD-ACE2 -binding inhibition in COVID- 19-positive and -negative samples where 0% is determined by the mean signal intensity of the healthy control samples (Fig 23D).

[0035] FIGURE 24 shows a lateral flow assay for sensitive and specific detection of anti- SARS-CoV-2 RBD specific, neutralising monoclonal antibodies, #37 and #42. The assay was performed using four anti-RBD monoclonal antibodies, two of which are known to be neutralising (Antibodies A and B), tested at serial 3-fold dilutions showing clear inhibition of signal at higher dilutions (indicated by molar ratios of antibodies to RBD protein). Two non-neutralising anti-RBD antibodies (Antibodies 1 and 2) did not prevent signal formation as expected. Incubation with increasing amounts of neutralising antibodies A and B (#37 and #42, respectively) resulted in inhibition of signal formation, indicating their ability to the interaction between RBD and ACE2 (Figure 24A; mAb #37 and #42 represented by the diamond-shaped data points). The signal intensity was quantified, as shown in Figure 24B and C. The degree of inhibition was surprisingly strong, even when the antibodies were present at only 0.11 molar ratio. This may reflect a level of inactive RBD protein that neither binds antibody nor ACE2. Another replicate is shown in Figure 24C.

[0036] FIGURE 25 shows a schematic depicting our lateral flow assay design (A). A photograph of two lateral flow strips side by side, with the left one showing a red band when RBD-Au has been included in the flow (B). Titration of RBD-Au run at a range of concentrations across the strips (C).

[0037] FIGURE 26 (A) shows ten representative COVID-19 -ve and COVID-19 +ve plasma samples. (B) shows conversion of stripe intensity from each sample to a % inhibition based on the formula % inhibition = (1 - sample intensity / median intensity of COVID-19-ve samples) xlOO. Plotted as % inhibition on y axis. (C) shows readings from lateral flow strips were plotted against readings for same samples derived from microneutralisation assay (R²=0.72; p<0.0001).

[0038] FIGURE 27 shows a schematic (A) and a photograph (B) depicting our prototype lateral flow assay cartridge design. 6 representative healthy pre-COVID-19 samples and 6 representative COVID-19 patient samples as run on the prototype lateral flow cartridge (C). (D) shows conversion of stripe intensity from each sample to a % inhibition based on the formula % inhibition = (1 - sample intensity / median intensity of COVID- 19-ve samples) xlOO. Readings from lateral flow strips were plotted against readings for same samples derived from a microneutralisation (MicroNeut titre) assay (E; R²=0.69; p<0.0001). (F) Samples from one individual that were collected from 3 to 10 weeks (C1.1-C1.5) and one individual at 4 weeks (C2.1). 6 COVID-19 -ve samples were run for comparison. (G). Data from patient C 1 (F) graphically presented as % inhibition versus days post symptom onset. [0039] FIGURE 28 shows (A) an N- and C-terminal truncated RBD of the original Wuhan- Hu-1 strain (SEQ ID NO:3) and three variant RBD (S477N; S477I; N439K) gold conjugates were titrated on ACE2 strips. Red line depicts direct binding between RBD and ACE2. Each variant showed a similar titration curve. (B) Samples from donor Cl were titrated against strips in the presence of Wuhan RBD or the 3 variants. (C) Data from B plotted to compare %inhibition against each different RBD.

[0040] FIGURE 29 shows (A) Schematic of the Version la LFA device; (B) Test strips performed with whole blood samples spiked with varying amounts of neutralising mAb#42, with the measured % inhibition calculated using the Axxin AX-2X data shown below each test strip; (C) Titration curve of % inhibition versus the amount of mAb#42, showing good correlation when a neutralizing antibody is titrated in a single whole blood sample; (D) Test strips performed with different COVID-19 neutralizing antibody positive clinical plasma samples spiked with red blood cells, with the neutralising antibody titre (ID50) shown above each test strip and the % inhibition from the lateral flow assay shown below each test strip.. [0041] FIGURE 30 shows (A) Schematic of the Version lb LFA device, (B) Test strips performed with whole blood samples without added antibody, or whole blood (WB), plasma or running buffer (RB) spiked with 10 pg/ml of neutralising mAb #42.

[0042] FIGURE 31 shows (A) a Version 2 LFA device, with the conjugate and anti- glycophorin at the lower part of the strip, equivalent to the sample pad of Version la and Version lb, and the whole blood (WB) or plasma sample and running buffer (RB) added sequentially to the same well. (B) a Version 3 device, with the conjugate and anti- glycophorin at the upper part of the strip before the nitrocellulose test component, equivalent to the position of the conjugate pad of Version la and Version lb, and the whole blood (or plasma) sample and buffer added sequentially to well A and well B. In both Versions 2 and 3, there is an additional 10 mm of Ahlstrom 8951 sample pad with anti-glycophorin A, after the sample application pad with both anti-glycophorin A and RBD-biotin-gold complex, to allow greater retention of red blood cells from 30 pl of whole blood.

[0043] FIGURE 32 shows (A) Version lb, as previously shown in Figure 30; (B) Version 2, showing equivalent levels of inhibition for mAb #42 spiked in each sample; and (C) Version 3, showing equivalent levels of inhibition for mAb #42 spiked in each sample.

[0044] FIGURE 33 shows (A) test strips in which plasma samples from patients Cl and C2 (collected at different time points) were mixed with an equal volume of packed red blood cells (RBC) to approximate the composition of whole blood. Samples (30 pl total, 15 pl plasma) were then analysed on the Version 2 UFA device. Axxin AX-2X results are tabulated below the individual test strips. (B) test strips in which only plasma samples were tested (15 pl total); (C) Graphical representation of the results from test strips shown in (A) and (B).

[0045] FIGURE 34 shows ACE2 binding inhibition in an LFA format using either the RBD antigen of SARS-CoV-2 (A), or a full-length trimeric form of the SARS-CoV-2 S protein (FHA) (B). Plasma from patient Cl at different time points, or patient C2 at a single time point, were tested on the two LFA devices. Negative control (Neg) is the absence of neutralising anti-SARS-CoV-2 antibodies. Positive control (Pos) is a SARS-CoV-2 neutralising monoclonal antibody (CB6; Shi et al., 2020; Nature, 584: 120-124).

[0046] FIGURE 35 is a quantitative representation of the Axxin AX-2X reader scans of the images shown in Figure 34.

[0047] FIGURE 36 is an assessment of neutralizing antibody titres in macaques immunised with SARS-CoV-2 spike protein. Three macaques were primed with spike protein vaccine in combination with Addavax™ adjuvant, and boosted with soluble RBD protein on day 21 (A). Eight additional macaques were primed with whole SARS-CoV-2 spike protein (day 0) and boosted with spike protein (day 21) (B). Blood samples were taken prior to the prime (day 0), post-prime (day 14; five animals only), prior to the boost (day 21), and post-boost (day 42). A-B depict, from left to right: raw Axxin readings; % inhibition (relative to prebleed baseline for each animal); and total anti-RBD antibody titre.

DETAILED DESCRIPTION

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Any materials and methods similar or equivalent to those described herein can be used to practice the present invention.

[0049] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0050] The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.

[0051] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0052] By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of . Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0053] As used herein the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a single antibody, as well as two or more antibodies; reference to "a tracer antigen" includes one tracer antigen molecule, as well as two or more molecules of the tracer antigen; and so forth.

[0054] As used herein, the term "about" refers to approximately a +/-10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0055] The present invention is predicated, at least in part, on the inventors' surprising finding that a lateral flow immunoassay (LFA) can be deployed to detect the presence of neutralising anti-viral antibodies in a sample, advantageously allowing the LFA to determine whether a subject, from whom the sample was collected, is likely to have developed at least some level of protective immunity against a virus. [0056] Thus, in an aspect disclosed herein, there is provided a lateral flow assay device for detecting an anti-viral antibody in a biological fluid sample, the device comprising, in the direction of flow:

(a) a sample application region, wherein the sample application region is configured to receive a biological fluid sample;

(b) a conjugation region comprising a tracer antigen, wherein the tracer antigen comprises a detectable moiety; and

(c) a test region comprising an immobilised capture antigen; wherein the device is configured to move the biological fluid sample by capillary action across the device in the direction of flow from the sample application region to the test region; wherein the tracer antigen or the immobilised capture antigen is a viral antigen or a functional variant thereof; and wherein

Lateral flow assay (LFA) device

[0057] Unless stated otherwise, the terms "lateral flow assay", "LFA", "lateral flow immunoassay" and "LFIA" are used interchangeably herein to denote an assay format or device that includes a series of operably connected active regions or elements at which various components of the assay are located. A common type of lateral flow assay device includes a zone, area or region for receiving the liquid sample, a conjugate region, and a reaction or test region. These assay devices are commonly known as lateral flow test strips and typically employ a porous material, e.g., nitrocellulose, defining a path for fluid flow and capable of supporting capillary flow. Other illustrative examples of suitable test strip material include Polyethylene terephthalate (PET) fibers, such as Dacron™ fibers, nitrocellulose, polyester, nylon, cellulose acetate, polypropylene, glass fibers, and a combinations of any of the foregoing materials and their backings.

[0058] Suitable LFA formats and devices, including immunochromatographic LFA, will be well known to persons skilled in the art, illustrative examples of which are described in the literature; see, e.g., "Rapid Lateral Flow Test Strips. Considerations for Product Development," EMD Millipore 2013; Koczula, etal., 2016, "Lateral flow assays," Essays in Biochemistry 60: 111-120; Sharma, et al., 2015, "Point-of-Care Diagnostics in Low Resource Settings: Present Status and Future Role of Microfluidics," Biosensors 5: 577-601; Sajid, et al., 2014, "Designs, formats and applications of lateral flow assay: A literature review," J. Saudi Chem. Soc. 19:689-705; Holstein, etal., "Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers," 2016, Analytical and Bioanalytical Chemistry 408(5): 1335-46; US Patent No. 9,034,657, "Two step lateral flow assay methods and devices"; US Patent Nos. 4,313,734; 4,376,110; 4,435,504; 4,703,017; 4,855,240; 4,954,452; 5,028,535; 5,075,078; 5,654,162; WO 95/16207; EP 0810436; and US Patent No. 8,859,265, "Lateral flow immunoassay device with a more rapid and accurate test result", each document incorporated by reference herein in its entirety. Other illustrative examples of suitable LFA formats are described in US Patent Nos. 5,559,041, 5,714,389, 5,120,643, and 6,228,660 and international publication nos. WO 2003/103835, WO 2005/089082, WO 2005/118139, and WO 2006/137785, each of which are incorporated herein by reference in their entirety.

[0059] Without being bound by theory or a particular mode of application, the general principle behind LFA can be described as a fluid sample (or an extract thereof) comprising the analyte(s) of interest flows (e.g., by capillary action) through various regions of a polymeric strip on which reagents are bound, immobilised or otherwise attached that can interact with the analyte(s) of interest. A typical lateral flow test strip may comprise overlapping membranes that are mounted on a substrate such as a backing card for better stability and handling. The fluid sample is applied at one end of the strip, on the adsorbent sample pad or application region, which is typically impregnated with buffer salts and surfactants that make the sample suitable for interaction with the detection system. The sample region ensures that the analyte(s) present in the sample are capable of binding to the capture reagents that are employed on the strip. The sample then flows or migrates through a conjugate release pad or conjugation region, which will comprise a binding moiety that is specific to the analyte(s) or interest. The binding moiety (also referred to herein as a tracer antigen) will suitably comprise a detectable moiety, typically a coloured or fluorescent molecule or particle, such as colloidal gold or latex microspheres. The sample, together with a complex comprising the binding moiety bound to the analyte(s) of interest, migrates along the strip towards the detection zone or test region. The detection zone / test region is typically a porous membrane (usually composed of nitrocellulose) comprising an immobilised capture probe (usually an antibody or a binding molecule). The capture probe is typically immobilised onto the test strip in a line configuration, although alternative configurations may be suitable. The immobilised capture probe reacts with the complex comprising the binding moiety and the analyte(s) of interest. Recognition of the sample analyte(s) results in a detectable response (e.g., change in colour) within the test region. An LFA device may optionally comprise a control region beyond the test region in the direction of flow. The control region will suitably comprise a binding agent that reacts with or binds to the binding moiety from the sample or the conjugation region, as an indicator that the device has performed properly, insofar as there has been sufficient flow of the sample and reagents from the sample application region across the strip to the control region. The read-out, typically represented by visible lines on the test strip, can be assessed by eye or by an automated reader. In an embodiment, the presence of an anti-viral antibody in the sample is indicated by the absence of a detectable signal visible to the naked eye at the test region.

[0060] Where an automated reader is used to detect a signal at the test and / or control regions, the optical density (OD) values can be used in combination with a pre-determined standard curve (calibration curve) to determine the concentration of the analyte(s) of interest in the sample.

[0061] LFA can be characterised into two general formats: direct and competitive. A direct format is typically used for larger analytes such as the p24 antigen used in the human immunodeficiency virus (HIV) test, as well as analytes with multiple antigenic sites, such as human chorionic gonadotropin (hCG), as used in pregnancy tests. The hCG test is an example of a sandwich-based assay, where the target is immobilized between two complementary antibodies. In the direct test, the presence of a visible test region indicates a positive result and the control region usually contains species-specific anti-immunoglobulin antibodies, specific for the antibody in the conjugate. In the case of small molecules with single antigenic determinants, which are unable to bind to two antibodies simultaneously, competitive LFA formats are often used.

[0062] The LFA format described herein is an example of a competitive LFA format, where the presence of the analyte(s) of interest (anti-viral antibodies) in the sample compete for binding to the viral antigen with either the tracer antigen or the immobilised capture antigen, thereby blocking, reducing, abrogating or otherwise inhibiting the interaction of the tracer antigen with the immobilised capture antigen at the test region. Accordingly, a positive result is shown by a weaker signal, or a lack of signal, at the test region, when compared to the signal that would otherwise be achieved in the absence of the analyte(s), while the control region should be visible independently of the test result.

[0063] In an embodiment, the LFA device may be configured to test multiple analytes simultaneously, including under the same conditions. For example, additional test regions of capture antigens specific to different analytes can be immobilized in an array format on the device. Examples of multiplex LFA formats are described elsewhere herein.

[0064] In an embodiment, multiple test regions comprising the same capture antigen in different amounts can be used for semi-quantitative analysis of the target analyte. The principle of this assay format is based on the stepwise capture of analyte-tracer antigen complexes by the immobilized capture antigen on each successive test region, where the number of detectable / visible regions appearing on the test strip is directly proportional to the concentration of the analyte in the sample.

[0065] As noted elsewhere herein, the sample will flow across the device, typically by capillary forces, from the sample application region to the control region. To maintain flow, an absorbent pad may be attached to the LFA device beyond the control region in the direction of flow, the purpose of which includes to wick any excess reagents and prevent backflow of the fluid along the device.

[0066] The term "direction of flow" is used herein to denote the flow of the fluid sample from the application region towards the control region of the LFA device. The flow is typically facilitated by capillary forces. The capillary driven flow may be advantageously controlled by one or more of methods suitable for interrupting the capillary flow of the fluid sample, illustrative examples of which include opening or closing an external vent, imposition of a soluble membrane along the flow path, imposition of a non-soluble but removable membrane along the flow path, decreasing the capillary force by compressing the capillary bed and limiting the flow path. Such devices can be incorporated into the LFA device during manufacture. Illustrative examples of sample flow control that can be employed in the LFA devices described herein are shown in US Patent Nos: 5,620,657; 5,705,397; 6,901,963; 7,803,319 and US patent publication Nos: 2002/0119486; 2010/0159599; 2011/0306072, the contents of which are incorporated herein by reference in their entirety.

[0067] The LFA device can be of any suitable shape and / or dimension, such as one or a combination of square, round, oval, polygonal, hexagonal, and the like. In an embodiment, the LFA device has a substantially rectangular shape or configuration.

[0068] The LFA device may suitably comprise a substrate comprising, at least in part, any bibulous or non-bibulous material, such as nitrocellulose, nylon, paper, glass fiber, dacron, polyester, polyethylene, olefin, or other cast or thermoplastic materials such as polyvinyl chloride, polyvinyl acetate, copolymers of vinyl acetate and vinyl chloride, polyamide, polycarbonate, polystyrene, etc. In an embodiment, at least one test strip material is nitrocellulose having a pore size of at least about 1 micron, more preferably of greater than about 5 microns, or about 8-12 microns. Suitable nitrocellulose sheets having a nominal pore size of up to approximately 12 microns, are available commercially from sources that will be known to persons skilled in the art, including, for example, Schleicher and Schuell GmbH. [0069] The LFA device may optionally include indicia that can include a designation for the test to be performed using the test strip. Such indicia may be printed on the test strip material using methods known in the art. Alternatively, indicia may be on other thin members, such as plastic or paper that are attached to the test strip, such as by adhesives, tape or the like.

[0070] The LFA device may include one or more materials. If a device comprises more than one material, the two or more of the materials are preferably in fluid communication with one another. For example, one material of the device may be overlaid on another material of the device, such as, for example, filter paper overlaid onto nitrocellulose. Alternatively, or in addition, the device may include a region comprising one or more materials followed by a region comprising one or more different materials. In this context, the regions will suitably be in fluid communication and may or may not partially overlap one another.

[0071] As noted elsewhere herein, the material or materials of the LFA device can be bound to a support or solid surface such as found, for example, in thin-layer chromatography and may have an absorbent pad either as an integral part or in liquid contact. For example, the device may comprise nitrocellulose sheet "backed", for example with a supporting sheet, such as a plastic sheet, to increase its handling strength. This can be manufactured by forming a thin layer of nitrocellulose on a sheet of backing material. The actual pore size of the nitrocellulose when backed in this manner will tend to be lower than that of the corresponding unbacked material. Alternatively, a pre-formed sheet of nitrocellulose and/or one or more other bibulous or non-bibulous materials can be attached to at least one supporting sheet, such as a sheet made of polymers (see, e.g., US Patent No. 5,656,503, the contents of which is incorporated herein by reference in its entirety). A supporting sheet can be transparent, translucent or opaque. Where the support sheet is transparent, the supporting sheet is preferably moisture impervious but can be moisture resistant or moisture pervious. In an embodiment, the device can be viewed through a window. In some embodiments, the window may comprise a transparent material such as glass, plastic or mylar.

[0072] The component parts of the LFA device may be present in a suitable housing, examples of which will be known to persons skilled in the art. The housing may suitably be configured to enclose the bibulous member and other assay components. The housing may be fabricated from any suitable material, where the material may be a material that is sufficiently rigid to maintain the integrity of the bibulous member and other components housed therein and will also be suitably inert to the various fluids and reagents that contact the housing during use. Suitable housing material includes plastics. The housing may include a port or analogous structure configured to allow sample application to the sample application region and one or more windows configured to allow viewing of the test and control regions. The housing may further comprise markings, such as test region and control region markings (e.g., "T" and "C"), etc.

[0073] The LFA device and methods disclosed herein may provide qualitative or quantitative results. Qualitative results typically include results that provide a simple "yes" or "no" determination of whether the analyte of interest (anti-viral antibody) is present in the sample being assayed. Qualitative results also include results that are positive if the amount of analyte in the sample exceeds a pre-determined threshold or reference value.

[0074] In an embodiment, where LFA device is configured to provide a qualitative result, such as where the analyte needs to be at a certain minimum concentration to be used in subsequent procedures, the assay device may be configured to have lower sensitivity than a comparable LFA device that is configured to detect the presence of the analyte(s) of interest at any concentration. Thus, in circumstances where a qualitative result in the format of an analyte simply being present in an amount that exceeds a pre-determined threshold is desired, the LFA device may be configured to have a sensitivity that is not sufficient to provide detection below the threshold. If the LFA device is too sensitive, there is a risk of a false positive result where an analyte that is too low in concentration to be useful nonetheless yields a positive result. This sensitivity can be set to any minimum amount of analyte in the sample. In some embodiments, multiple LFA devices (e.g., in the form of test strips) may be supplied (e.g., in the form of a kit) with different sensitivities depending on the necessary threshold for analyte utility. These types of qualitative embodiments are suitably distinguished from LFA devices that are configured to be sensitive for all levels of analyte in a given sample. The desired sensitivity may be provided in a given LFA device using any convenient protocol, such as by providing an appropriate amount of capture agent in the detection region, etc.

[0075] In contrast, quantitative results provide some measurement of how much of the analyte(s) of interest is present in the sample. Accordingly, a quantitative result provides at least an approximation of the amount of the analyte(s) of interest that is present in the sample being assayed. To provide for quantitative results, the detection region may suitably include two or more distinct test regions that include the same or different amounts of the same capture antigen. As such, if the amount of analyte in the sample exceeds the amount of the analyte that can be captured in the first test region, the remaining free analyte will move to the second or subsequent test region. The resultant positive results from the first and second or subsequent test regions provide a quantitative measurement of the amount of analyte in the sample. By having a series of regions, which may be a gradient of two or more test regions each having differing (such as decreasing) amounts of capture antigen, a quantitative measurement of the analyte in the sample may be obtained. Alternatively, quantitative measurements can be obtained by densitometry, where only one capture region may be sufficient.

[0076] The present disclosure also extends to a multiplex LFA format in which the presence of two or more distinct (i.e., different) analytes (e.g., that differ from each other by their binding specificity for a viral antigen of interest) in the sample is determined, either qualitatively or quantitatively. The number of distinct analytes that may be detected in a given multiplex assay may vary, ranging in some instances from 2 to at least 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and so on). To provide for multiplex analysis, the configuration of the lateral flow assay device may vary. For example, the lateral flow assay device may include a single sample application region and a test region that includes capture antigens for each of the two or more analytes, where the different capture antigens may be present in the same test region or in different test regions, which may depend on whether the detectable moieties employed for each analyte are distinguishable from each other. Accordingly, the lateral flow assay device may suitably include a single flow lane linking the sample application region to the test region. Another example of a suitable multiplex configuration includes a separate sample application region and test region for each of the two or more analytes of interest. Accordingly, the lateral flow assay device may include two or more distinct flow lanes, each having its own sample application region and test region. Other configurations include a configuration having multiple flow lanes extending from a single sample application region to multiple test regions, where a separate test region is provided for each analyte of interest. Additional details regarding multiplex configurations are described in US Patent No. 6,037,127, the disclosure of which is herein incorporated by reference in its entirety. Thus, in an embodiment disclosed herein, the LFA device disclosed herein comprises an array or a plurality (e.g., 2, 3, 4, 5 and so on) of test strips configured to detect a plurality of analytes of interest. For example, the LFA device may comprise a plurality of test strips configured to (i) receive the sample (whether via a single sample application region or a plurality of sample application regions) and (ii) move the sample by capillary action in the direction of flow from the sample application region(s) to each of the plurality of test strips, wherein at least two of the plurality of test strips each comprises a test region for detecting the presence of a different neutralizing anti-viral antibody. Such multiplex configurations may suitably be used to detect neutralizing antibodies against different virus strains. For example, the LFA device may comprise a first test strip configured to detect Wuhan-Hu- 1 neutralizing antibodies in a sample, and a second test strip configured to detect the presence of neutralizing antibodies of a variant strain of SARS- CoV-2 in the sample. Alternatively, or in addition, the LFA device may comprise a test strip configured to detect the presence of neutralizing antibodies to other viruses (e.g., to an influenza virus). The array or plurality of test strips may suitably be configured in the same cassette or housing, whether in a parallel configuration or otherwise, for ease of use. Illustrative examples of multiplex LFA devices are described in Anfossi et al. (2019, Biosensors (Basel),' 9(1):2). Thus, in an embodiment disclosed herein, there is provided a multiplex LFA device comprising:

(a) a first LFA test strip configured to detect the presence of neutralizing antibodies to a first viral antigen or a functional variant thereof; and

(b) a second LFA test strip configured to detect the presence of neutralizing antibodies to a second viral antigen or a functional variant thereof, wherein the first and second LFA test strips are configured in the same cassette or housing. [0077] In an embodiment, the first and second LFA test strips of the multiplex LFA device are configured to receive the same biological fluid sample.

[0078] In an embodiment, the LFA formats and devices described herein may be suitably are deployed as one step in a multi-step research protocol, where the protocol at least includes a further step, either before or after the step of analyte detection, as described herein. Thus, the present disclosure extends to a research protocol that includes a first step, an analyte detection step and then a subsequent step. For example, the methods disclosed herein include a step of preparing the sample, a step of testing the sample for the analyte of interest and then a step of further using the sample in a research procedure, e.g., a further method performed in a laboratory.

[0079] The present disclosure relates generally to an LFA device and uses thereof for determining the presence of anti-viral antibodies in a sample; that is, antibodies that specifically bind to a viral antigen. Where the fluid sample is derived from a subject, the LFA device may suitably detect the presence of anti-viral antibodies the subject has generated in response to prior exposure to the virus (e.g., a prior viral infection) and / or to the viral antigen (e.g., following immunisation). The LFA devices and methods described herein advantageously allow for the detection of neutralising anti-viral antibodies in the sample, including a fluid sample from a subject. In this context, the term "neutralising" is not intended to mean that the subject has developed an anti-viral antibody titre as a result of prior exposure to the virus or to a viral antigen (e.g., following immunization) that is sufficient to provide complete protection against subsequent infection by the virus. Rather, the term "neutralising" is intended to mean that the anti-viral antibodies are capable of binding to a viral antigen of the native virus and thereby inhibit the interaction of the virus with its receptor that would otherwise facilitate entry of the virus into a cell. For example, neutralising anti-influenza A antibodies will bind to influenza A and at least partially inhibit the binding of the virus to hemagglutinin (HA). By way of further example, neutralising anti-SARS-CoV-2 antibodies will bind to SARS-CoV-2 and at least partially inhibit the binding of the virus to its known receptor, Angiotensin Converting Enzyme 2 (ACE2).

[0080] The reagents and components of the LFA device can be described by reference to different regions of the device. Using the guidance provided herein, the knowledge of one of skill in the art and the published literature, reagents and components for use in each region can be selected and incorporated into the device and the assay format on the basis of individual needs, available resources, etc, as appropriate.

[0081] In some embodiments, the LFA device is configured to detect anti-viral antibodies to more than one viral protein. For instance, the LFA device may be configured to detect antibodies to two or more a viral proteins of the same virus. Alternatively, or in addition, the LFA device may be configured to detect anti-viral antibodies to two or more viral antigens, wherein each of the two or more viral antigens are from different viruses. The multiplex approach advantageously allows multiple anti-viral antibodies to be detected in a single assay, thereby reducing the time and costs that would otherwise be spent performing multiple assays. Where the LFA device is configured to detect anti-viral antibodies to two or more viral antigens, the tracer antigen for each analyte may suitably carry a different detectable moiety so that a result (i.e., the presence or absence of the analyte and the control signal) can be differentiated between analytes / tracer antigens. Alternatively, or in addition, the LFA device can be configured to detect each of the two or more anti-viral antibodies at different test regions. Alternatively, or in addition, the LFA device can also be configured to detect each of the two or more tracer antigens at different control regions.

Sample application region

[0082] A sample application region is a region of the LFA device that is adapted to receive a sample that is to be assessed for the presence of anti-viral antibodies. A sample application region can include a bibulous or non-bibulous material, such as filter paper, nitrocellulose, glass fibres, polyester or other appropriate materials known to persons skilled in the art. One or more materials of the sample application region may perform a filtering function, such that large particles or cells are prevented from moving through the test strip. A sample application region can be in direct or indirect fluid communication with the remainder of the LFA device, including the conjugation, test and control regions. A direct or indirect fluid communication can be, for example, end-to-end communication, overlap communication, or overlap or end-to-end communication that involves another element, such as a fluid communication structure such as filter paper.

[0083] A sample application region may optionally include compounds or molecules that may be necessary or desirable for optimal performance of the assay. Such compounds or molecules will be familiar to persons skilled in the art, illustrative examples of which include one or more of added, pre-added or post-added buffers, stabilizers, surfactants, salts and reducing agents.

[0084] It is to be understood that the LFA device disclosed herein can be used to detect the presence of anti-viral antibodies in any fluid sample that contains, or is expected to contain, anti-viral antibodies. Suitable fluid samples will be familiar to persons skilled in the art, illustrative examples of which include biological fluid samples (e.g., blood, serum, plasma, saliva, urine, saliva, nasopharyngeal secretion) and culture media derived from cultures of cells that produce anti-viral antibodies, including hybridomas.

[0085] The term "sample", as used herein, means a volume of a liquid, solution or suspension, intended to be subjected to qualitative or quantitative determination of the presence or absence of the analyte(s) of interest (i.e., anti-viral antibodies), the concentration of the analyte(s) of interest, etc. Illustrative examples of suitable samples include human or animal bodily fluids, such as blood, plasma, serum, lymph, urine, saliva, semen, amniotic fluid, gastric fluid, phlegm, sputum, mucus, nasopharyngeal secretion, tears, stool, etc. Other examples of suitable samples are derived from human or animal tissue samples where the tissue sample has been processed into a liquid, solution, or suspension to reveal particular tissue components for examination. The LFA device described herein may advantageously be configured to receive a sample of whole blood. In this context, the LFA device may conveniently be used as a point-of-care device that requires little or no prior sample processing. Thus, in an embodiment, the sample is a blood sample.

[0086] In other embodiments, the sample may suitably be processed before being applied to the sample application region of the LFA device, for example, to remove at least some of the non-antibody products or impurities that may be found in the sample (e.g., cells, nonimmunoglobulin proteins, etc), thereby producing a sample that is at least partially enriched for antibodies. Suitable methods of processing a sample prior to being applied to the sample application region will be familiar to persons skilled in the art, illustrative examples of which are described elsewhere herein and include centrifugation of whole blood samples and extraction of the plasma from the cell fraction or exposing the sample to an immobilised binding agent that specifically binds to and removes cells, including red blood cells, from the sample (e.g., anti-glycophorin A antibodies). Alternatively, the fluid sample may be applied to the sample application region of the LFA device without prior processing, which is advantageous for point-of-care applications where sample processing is not available or would otherwise be too time consuming and expensive. In some embodiments, the sample application region is configured to process the sample subsequent to the sample being applied to the sample application region and prior to the sample moving from the sample application region to the test region. For example, the sample application region may suitably comprise a whole blood filter and the like, which aids in the separation of cells and, optionally, cellular debris, from the blood sample that may otherwise interfere with and reduce the performance of the assay. Suitable whole blood filters that may be configured into the LFA device will be familiar to persons skilled in the art, illustrative examples of which are described in Koczula and Gallotta (2016; 2016, 30; 60(1): 111-

120).

[0087] In an embodiment, the sample application region will suitably comprise chromatographic material. The sample application region may comprise a single chromatographic material, or several capillary active materials made of the same or different materials, preferably fixed onto a carrier backing. These materials will typically be in fluid communication with each other so as to form a transport path along which a sample driven by capillary forces flows from the sample application region, passing the conjugation region, towards one or more test regions and, optionally, an absorbent region at the other end of the device.

[0088] In an embodiment, the sample is directly applied to the LFA device by dipping the sample application region of the LFA device into the fluid sample. Alternatively, application of the sample to the LFA device may be carried out by collecting the sample with a dry or wetted wiping element from which the sample can be transferred, optionally after moistening, to the sample application region. The wiping element may suitably be sterile and may be dry or pretreated with a fluid before the collection step. Materials suitable for wiping elements may comprise synthetic materials, woven fabrics or fibrous webs. Illustrative examples of suitable wiping elements are described in German Patents DE 44 39 429 and DE 196 22 503, which are hereby incorporated by reference in their entirety. In other embodiments, the sample may be collected by a collection receptacle, such as a pipette, and transferred directly to the sample application region of the LFA device.

[0089] The volume of fluid sample required to be applied to the sample application region will be known to persons skilled in the art and will likely depend on the configuration of the LFA device and its components, including, for example, the distance between the sample application region and the test and / or control regions in the direction of flow, the capacity of the chromatographic material to carry the fluid sample and analytes from the sample application region to the test and / or control regions in the direction of flow, etc. In an embodiment, a suitable amount of whole blood applied to the sample application region is about 5 uL to about 50 uL. In an embodiment, a suitable amount of whole blood applied to the sample application region is at least about 5 uL. In an embodiment, a suitable amount of whole blood applied to the sample application region is at most about 50 uL. In an embodiment, a suitable amount of whole blood applied to the sample application region is about 5 uL, preferably about 10 uL, preferably about 15 uL, preferably about 20 uL, preferably about 25 uL, preferably about 30 uL, preferably about 35 uL, preferably about 40 uL, preferably about 45 uL, or more preferably about 50 uL. In an embodiment, a suitable amount of whole blood applied to the sample application region is about 5 uL to about 10 uL, about 5 uL to about 15 uL, about 5 uL to about 20 uL, about 5 uL to about 25 uL, about 5 uL to about 30 uL, about 5 uL to about 25 uL, about 5 uL to about 40 uL, about 5 uL to about 45 uL, about 5 uL to about 50 uL, about 10 uL to about 15 uL, about 10 uL to about 20 uL, about 10 uL to about 25 uL, about 10 uL to about 30 uL, about 10 uL to about 25 uL, about 10 uL to about 40 uL, about 10 uL to about 45 uL, about 10 uL to about 50 uL, about 15 uL to about 20 uL, about 15 uL to about 25 uL, about 15 uL to about 30 uL, about 15 uL to about 25 uL, about 15 uL to about 40 uL, about 15 uL to about 45 uL, about 15 uL to about 50 uL, about 20 uL to about 25 uL, about 20 uL to about 30 uL, about 20 uL to about 25 uL, about 20 uL to about 40 uL, about 20 uL to about 45 uL, about 20 uL to about 50 uL, about 25 uL to about 30 uL, about 25 uL to about 25 uL, about 25 uL to about 40 uL, about 25 uL to about 45 uL, about 25 uL to about 50 uL, about 30 uL to about 25 uL, about 30 uL to about 40 uL, about 30 uL to about 45 uL, about 30 uL to about 50 uL, about 25 uL to about 40 uL, about 25 uL to about 45 uL, about 25 uL to about 50 uL, about 40 uL to about 45 uL, about 40 uL to about 50 uL, or about 45 uL to about 50 uL. [0090] In an embodiment, a suitable amount of serum or plasma applied to the sample application region is about 3 uL to about 30 uL. In an embodiment, a suitable amount of serum or plasma applied to the sample application region is at least about 3 uL. In an embodiment, a suitable amount of serum or plasma applied to the sample application region is at most about 30 uL. In an embodiment, a suitable amount of serum or plasma applied to the sample application region is about 2 uL, preferably about 3 uL, preferably about 4 uL, preferably about 5 uL, preferably about 6 uL, preferably about 7 uL, preferably about 8 uL, preferably about 9 uL, preferably about 10 uL, preferably about 11 uL, preferably about 12 uL, preferably about 13 uL, preferably about 14 uL, preferably about 15 uL, preferably about 16 uL, preferably about 17 uL, preferably about 18 uL, preferably about 19 uL, preferably about 20 uL, preferably about 25 uL, preferably about 30 uL, preferably about 35 uL, preferably about 40 uL, preferably about 45 uL, or more preferably about 50 uL. In an embodiment, a suitable amount of serum or plasma applied to the sample application region is about 3 uL to about 5 uL, about 3 uL to about 8 uL, about 3 uL to about 10 uL, about 3 uL to about 13 uL, about 3 uL to about 15 uL, about 3 uL to about 18 uL, about 3 uL to about 20 uL, about 3 uL to about 23 uL, about 3 uL to about 25 uL, about 3 uL to about 28 uL, about 3 uL to about 30 uL, about 5 uL to about 8 uL, about 5 uL to about 10 uL, about 5 uL to about 13 uL, about 5 uL to about 15 uL, about 5 uL to about 18 uL, about 5 uL to about 20 uL, about 5 uL to about 23 uL, about 5 uL to about 25 uL, about 5 uL to about 28 uL, about 5 uL to about 30 uL, about 8 uL to about 10 uL, about 8 uL to about 13 uL, about 8 uL to about 15 uL, about 8 uL to about 18 uL, about 8 uL to about 20 uL, about 8 uL to about 23 uL, about 8 uL to about 25 uL, about 8 uL to about 28 uL, about 8 uL to about 30 uL, about 10 uL to about 13 uL, about 10 uL to about 15 uL, about 10 uL to about 18 uL, about 10 uL to about 20 uL, about 10 uL to about 23 uL, about 10 uL to about 25 uL, about 10 uL to about 28 uL, about 10 uL to about 30 uL, about 13 uL to about 15 uL, about 13 uL to about 18 uL, about 13 uL to about 20 uL, about 13 uL to about 23 uL, about 13 uL to about 25 uL, about 13 uL to about 28 uL, about 13 uL to about 30 uL, about 15 uL to about 18 uL, about 15 uL to about 20 uL, about 15 uL to about 23 uL, about 15 uL to about 25 uL, about 15 uL to about 28 uL, about 15 uL to about 30 uL, about 18 uL to about 20 uL, about 18 uL to about 23 uL, about 18 uL to about 25 uL, about 18 uL to about 28 uL, about 18 uL to about 30 uL, about 20 uL to about 23 uL, about 20 uL to about 25 uL, about 20 uL to about 28 uL, about 20 uL to about 30 uL, about 23 uL to about 25 uL, about 23 uL to about 28 uL, about 23 uL to about 30 uL, about 25 uL to about 28 uL, about 25 uL to about 30 uL, or about 28 uL to about 30 uL.

[0091] In an embodiment, a suitable amount of saliva applied to the sample application region is about 5 uL to about 50 uL. In an embodiment, a suitable amount of saliva applied to the sample application region is at least about 5 uL. In an embodiment, a suitable amount of saliva applied to the sample application region is at most about 50 uL. In an embodiment, a suitable amount of saliva applied to the sample application region is about 5 uL, preferably about 10 uL, preferably about 15 uL, preferably about 20 uL, preferably about 25 uL, preferably about 30 uL, preferably about 35 uL, preferably about 40 uL, preferably about 45 uL, or more preferably about 50 uL. In an embodiment, a suitable amount of saliva applied to the sample application region is about 5 uL to about 10 uL, about 5 uL to about 15 uL, about 5 uL to about 20 uL, about 5 uL to about 25 uL, about 5 uL to about 30 uL, about 5 uL to about 25 uL, about 5 uL to about 40 uL, about 5 uL to about 45 uL, about 5 uL to about 50 uL, about 10 uL to about 15 uL, about 10 uL to about 20 uL, about 10 uL to about 25 uL, about 10 uL to about 30 uL, about 10 uL to about 25 uL, about 10 uL to about 40 uL, about 10 uL to about 45 uL, about 10 uL to about 50 uL, about 15 uL to about 20 uL, about 15 uL to about 25 uL, about 15 uL to about 30 uL, about 15 uL to about 25 uL, about 15 uL to about 40 uL, about 15 uL to about 45 uL, about 15 uL to about 50 uL, about 20 uL to about 25 uL, about 20 uL to about 30 uL, about 20 uL to about 25 uL, about 20 uL to about 40 uL, about 20 uL to about 45 uL, about 20 uL to about 50 uL, about 25 uL to about 30 uL, about 25 uL to about 25 uL, about 25 uL to about 40 uL, about 25 uL to about 45 uL, about 25 uL to about 50 uL, about 30 uL to about 25 uL, about 30 uL to about 40 uL, about 30 uL to about 45 uL, about 30 uL to about 50 uL, about 25 uL to about 40 uL, about 25 uL to about 45 uL, about 25 uL to about 50 uL, about 40 uL to about 45 uL, about 40 uL to about 50 uL, or about 45 uL to about 50 uL.

[0092] In an embodiment, a suitable amount of urine applied to the sample application region is about 5 uL to about 50 uL. In an embodiment, a suitable amount of urine applied to the sample application region is at least about 5 uL. In an embodiment, a suitable amount of urine applied to the sample application region is at most about 50 uL. In an embodiment, a suitable amount of urine applied to the sample application region is about 5 uL, preferably about 10 uL, preferably about 15 uL, preferably about 20 uL, preferably about 25 uL, preferably about 30 uL, preferably about 35 uL, preferably about 40 uL, preferably about 45 uL, or more preferably about 50 uL. In an embodiment, a suitable amount of urine applied to the sample application region is about 5 uL to about 10 uL, about 5 uL to about 15 uL, about 5 uL to about 20 uL, about 5 uL to about 25 uL, about 5 uL to about 30 uL, about 5 uL to about 25 uL, about 5 uL to about 40 uL, about 5 uL to about 45 uL, about 5 uL to about 50 uL, about 10 uL to about 15 uL, about 10 uL to about 20 uL, about 10 uL to about 25 uL, about 10 uL to about 30 uL, about 10 uL to about 25 uL, about 10 uL to about 40 uL, about 10 uL to about 45 uL, about 10 uL to about 50 uL, about 15 uL to about 20 uL, about 15 uL to about 25 uL, about 15 uL to about 30 uL, about 15 uL to about 25 uL, about 15 uL to about 40 uL, about 15 uL to about 45 uL, about 15 uL to about 50 uL, about 20 uL to about 25 uL, about 20 uL to about 30 uL, about 20 uL to about 25 uL, about 20 uL to about 40 uL, about 20 uL to about 45 uL, about 20 uL to about 50 uL, about 25 uL to about 30 uL, about 25 uL to about 25 uL, about 25 uL to about 40 uL, about 25 uL to about 45 uL, about 25 uL to about 50 uL, about 30 uL to about 25 uL, about 30 uL to about 40 uL, about 30 uL to about 45 uL, about 30 uL to about 50 uL, about 25 uL to about 40 uL, about 25 uL to about 45 uL, about 25 uL to about 50 uL, about 40 uL to about 45 uL, about 40 uL to about 50 uL, or about 45 uL to about 50 uL.

Conjugation region and tracer antigens

[0093] The conjugation region will suitably comprises a tracer antigen, configured such that the tracer antigen will migrate with the sample (and with the analyte(s) of interest, if present in the sample) towards the test and control regions of the LFA device in the direction of flow. As noted elsewhere herein, the present inventors have surprisingly found that neutralising anti-viral antibodies can be detected in a sample by employing a competitive LFA format. In the absence of anti-viral antibodies in the sample, a complex comprising the tracer antigen from the conjugation region and the capture antigen at the test region is formed to produce a detectable test signal at the test region. Conversely, in the presence of an antiviral antibody in the sample, the anti-viral antibody inhibits the formation of the complex comprising the tracer antigen from the conjugation region and the capture antigen at the test region, thereby producing a weaker detectable test signal at the test region when compared to the detectable test signal that would otherwise be produced at the test region in the absence of the anti-viral antibody in the biological fluid sample. Thus, the tracer antigen is suitably paired with the capture antigen to be employed in the test region, such that the tracer antigen and the capture antigen are capable of forming a complex at the test region in the absence of neutralising an anti-viral antibodies so as to produce a detectable signal at the test region. For example, where the tracer antigen is a viral antigen, or a functional variant thereof, the immobilised capture antigen may be a receptor of the viral antigen, or an antigen-binding variant thereof. Conversely, where the tracer antigen is a virus receptor, or a functional variant thereof, the immobilised capture antigen will suitably be a viral antigen, or a functional variant thereof, to which the virus receptor or variant is capable of binding.

[0094] Suitable tracer antigens (and paired captured antigens) will be familiar to persons skilled in the art, illustrative examples of which include viral antigens, virus receptors and functional variants thereof, such as those described elsewhere herein. In an embodiment, the tracer antigen is a viral antigen or a functional variant thereof, as described herein. In another embodiment, the tracer antigen is a virus receptor, or a functional variant thereof, as described herein.

[0095] The tracer antigen suitably comprises a detectable moiety that enables the tracer antigen to be detected in the test and control regions of the LFA device.

[0096] The choice of detectable moiety may vary widely depending on the LFA format and / or device and may be any directly or indirectly detectable label. Suitable detectable moieties for use in the devices and methods disclosed herein thus include any moiety that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other means. Suitable detectable moieties will be familiar to persons skilled in the art, illustrative examples of which include metal particles, such as gold, or polymeric beads, such as coloured beads, particles of carbon black, biotin, streptavidin and any combination of the foregoing. Other illustrative examples of suitable detectable moieties include enzymes, chromophores and fluorophores. In an embodiment, the detectable moiety is selected from the group consisting of colloid gold, biotin, streptavidin and any combination of the foregoing. In an embodiment, the detectable moiety comprises colloid gold. The tracer antigen may suitably comprise two or more detectable moieties, for instance, where a stronger detectable signal is required (e.g., for greater sensitivity where the level of analyte in the sample is expected to be low). Suitable combinations of two or more detectable moieties will be familiar to persons skilled in the art. In an embodiment, the detectable moiety comprises colloid gold and biotin. [0097] Other suitable detectable moieties include biotin for staining with labelled streptavidin conjugate, fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g.. ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels, such as colloidal gold or coloured glass or plastic (e.g., polystyrene, polypropylene, latex beads). Illustrative examples of suitable detectable moieties are describe in US Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275, 149 and 4,366,241. See also Handbook of Fluorescent Probes and Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene Oreg.). Radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colourimetric labels are detected by simply visualizing the coloured label.

[0098] The LFA device may suitably include one or more substrates that change in an optical property (such as colour, chemiluminescence or fluorescence) to produce a detectable signal when the tracer antigen binds to the immobilised capture antigen at the test region and when the tracer antigen binds to the capture reagent at the control region. Suitable substrates will be known to persons skilled in the art, illustrative examples of which include 1,2- phenylenediamine, 5-aminosalicylic acid, 3,3',5,5'tetra methyl benzidine, or tolidine for peroxidase; 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium for alkaline phosphatase and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, o-nitrophenyl- beta-D-galactopyranoside, napthol-AS-BI-beta-D-galactopyranoside, and 4-methyl- umbelliferyl-beta-D-galactopyranoside for beta galactosidase. The one or more substrates that produce the detectable signal can be provided elsewhere in the LFA device and can migrate to the test and control regions. Alternatively, or in addition, the one or more substrates that produce the detectable signal can suitably be provided at the test and control regions.

[0099] The conjugation region may optionally comprise one or more detectable components capable of being detected at a control region of the device (whether on the same device as the test region or a separate device, as described elsewhere herein). It will be understood that any suitable detectable component can be used, as long as it is capable of forming a detectable complex with the immobilised control agent at the control region to produce a detectable control signal. Thus, the detectable component will suitably be a binding partner to the immobilised control agent so as to enable the formation of a detectable complex comprising the detectable component and the immobilised control agent. Suitable detectable components will be familiar to persons skilled in the art, non-limiting examples of which include streptavidin, biotin and immunoglobulin molecules. In an embodiment, the detectable component may comprise an antibody or an antigen binding fragment thereof that specifically binds to immunoglobulin found in the sample. Suitable antibodies and antigen binding fragments thereof will be familiar to persons skilled in the art and may be speciesand / or isotype-specific.

Test region and immobilised capture antigens

[0100] The test region of an LFA device is typically a porous membrane (e.g., composed of nitrocellulose) with capture antigens immobilized thereon, typically in a line, spot or other suitable configuration. In the absence of the analyte(s) of interest, the immobilised capture antigen will react with the tracer antigen from the conjugation region to form a complex at the test region, which will suitably produce a detectable signal by virtue of the detectable moiety that is attached to the tracer antigen. The detectable signal, which can appear as a colour change at the test region, is typically assessed by eye or by using an automated reader. [0101] As noted elsewhere herein, the type of immobilised capture antigen at the test region will depend on the tracer antigen that is employed in the conjugation region of the LFA device, insofar as the immobilised capture antigen will necessarily comprises a structure or amino acid sequence that is capable of binding specifically to the tracer antigen to form a detectable tracer / capture antigen complex at the test region of the LFA device. In the absence of an anti-viral antibody in the sample, a complex comprising the tracer antigen and the capture antigen is formed to produce a detectable test signal at the test region, whereas in the presence of an anti-viral antibody in the sample, the anti-viral antibody inhibits the formation of the tracer antigen / capture antigen complex at the test region, thereby producing a weaker detectable test signal at the test region when compared to a detectable test signal produced in the absence of the anti-viral antibody in the sample. Thus, where the tracer antigen is a viral antigen, or a functional variant thereof, the immobilised capture antigen may suitably comprise a receptor for the viral antigen, or a viral antigen-binding variant thereof. Conversely, where the tracer antigen is a receptor for the viral antigen, or a viral antigen-binding variant thereof, the immobilised capture antigen may suitably comprise the viral antigen or a functional variant thereof. Suitable combinations or binding pairs of tracer antigens and capture antigens to be employed in the LFA device will be familiar to persons skilled in the art, having regard to the type of anti-viral antibodies the LFA device is to be used to detect. Thus, the immobilised capture antigen of the test region will suitably be paired with the tracer antigen that is to be employed in the conjugation region, such that the tracer antigen and the capture antigen are capable of forming a complex at the test region in the absence of the analyte(s) of interest (anti-viral antibodies) so as to produce a detectable signal at the test region.

[0102] As noted elsewhere herein, a complex comprising the tracer antigen and the capture antigen is formed at the test region of the LFA device in the absence of an anti-viral antibody in the fluid sample, thereby producing a detectable test signal at the test region. In the presence of an anti-viral antibody in the fluid sample, the anti-viral antibody inhibits the formation of the complex comprising the tracer antigen and the capture antigen at the test region, thereby producing a weaker detectable test signal at the test region when compared to a detectable test signal produced in the absence of the anti-viral antibody in the fluid sample. Thus, a positive result is shown by a weaker detectable signal when compared to a detectable test signal produced in the absence of the anti-viral antibody in the sample. Without being bound by theory or by a particular mode of application, the anti-viral antibody inhibits the formation of the complex comprising the tracer antigen and the capture antigen by (i) competing for binding to the tracer antigen, where the tracer antigen comprises, consists or consists essentially of a viral antigen or a functional variant thereof, as described herein, or (ii) competing for binding to the immobilised capture antigen, where the where the tracer antigen comprises, consists or consists essentially of a viral antigen or a functional variant thereof, as described herein.

[0103] In an embodiment, the immobilised capture antigen is a receptor for the viral antigen or a viral antigen-binding variant thereof, as described herein. Suitable viral antigens will be familiar to persons skilled in the art, illustrative examples of which are described elsewhere herein. In an embodiment, the immobilised capture antigen comprises, consists or consists essentially of an angiotensin converting enzyme 2 (ACE2) or a SARS-CoV-2 RBD-binding fragment thereof, as described herein. In an embodiment, the ACE2 is human ACE2. In an embodiment, the immobilised capture antigen comprises, consists or consists essentially of an angiotensin converting enzyme 2 (ACE2) or a SARS-CoV-2 RBD-binding variant thereof, and the tracer antigen comprises, consists or consists essentially of a SARS- CoV-2 viral protein or a functional variant thereof, as described elsewhere herein.

[0104] In another embodiment, the immobilised capture antigen comprises, consists or consists essentially of a SARS-CoV-2 viral protein or a functional variant thereof, as described elsewhere herein. In an embodiment, the immobilised capture antigen comprises, consists or consists essentially of a SARS-CoV-2 viral protein or a functional variant thereof, and the tracer antigen comprises, consists or consists essentially of an angiotensin converting enzyme 2 (ACE2) or a SARS-CoV-2 RBD-binding variant thereof, as described elsewhere herein. In an embodiment, the immobilised capture antigen comprises, consists or consists essentially of the SARS-CoV-2 RBD or a functional variant thereof, as described herein. In an embodiment, the immobilised capture antigen comprises, consists or consists essentially of the SARS-CoV-2 RBD or a functional variant thereof, as described herein, and the tracer antigen comprises, consists or consists essentially of an angiotensin converting enzyme 2 (ACE2) or a SARS-CoV-2 RBD-binding variant thereof, as described elsewhere herein.

[0105] The amount of capture antigen suitably immobilised at the test region of the LFA device may vary, depending on the sensitivity of the assay that may be attributed, at least in part, to factors such as the amount of sample to be applied to the device, the concentration of the analyte(s) of interest in the sample, the binding affinity of the analyte(s) of interest to the viral antigen(s) employed in the device, the amount of interfering molecules that may be present in the sample (i.e., molecules that may non-specifically interfere with and inhibit the interaction or binding of the analyte(s) of interest in the sample to the viral antigen(s), the binding affinity of the tracer antigen to the capture antigen, and the amount of interfering molecules that may be present in the sample that non-specifically interfere with and inhibit the interaction between the tracer antigen and the capture antigen. The amount of capture antigen immobilised at the test region can be optimised using standard trial and error that will be familiar to persons skilled in the art. In an embodiment, the capture antigen is immobilised at the test region in an amount in the range from about 0. 1 mg/mL to about 10 mg/mL, preferably from about 0.2 mg/mL to about 10 mg/mL, preferably from about 0.3 mg/mL to about 10 mg/mL, preferably from about 0.4 mg/mL to about 10 mg/mL, preferably from about 0.5 mg/mL to about 10 mg/mL, preferably from about 0.6 mg/mL to about 10 mg/mL, preferably from about 0.7 mg/mL to about 10 mg/mL, preferably from about 0.8 mg/mL to about 10 mg/mL, preferably from about 0.9 mg/mL to about 10 mg/mL, preferably from about 1 mg/mL to about 10 mg/mL, preferably from about 2 mg/mL to about 10 mg/mL, or preferably from about 2 mg/mL to about 10 mg/mL. In an embodiment, the capture antigen is immobilised at the test region in an amount of at least about 0. 1 mg/mL (i.e., 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 mg/mL, and so on), preferably at least about 0. 1 mg/mL, preferably at least about 0.5 mg/mL, preferably at least about 1.0 mg/mL, preferably at least about 1.5 mg/mL, preferably at least about 2.0 mg/mL, preferably at least about 2.5 mg/mL, preferably at least about 3.0 mg/mL, preferably at least about 3.5 mg/mL, preferably at least about 4.0 mg/mL, preferably at least about 4.5 mg/mL, preferably at least about 5.0 mg/mL, preferably at least about 5.5 mg/mL, preferably at least about 6.0 mg/mL, preferably at least about 6.5 mg/mL, preferably at least about 7.0 mg/mL, preferably at least about 7.5 mg/mL, preferably at least about 8.0 mg/mL, preferably at least about 8.5 mg/mL, preferably at least about 9.0 mg/mL, preferably at least about 9.5 mg/mL, or more preferably at least about 10 mg/mL.

[0106] In an embodiment, the capture antigen is a SARS-CoV-2 viral protein, or a functional variant thereof, as described herein, immobilised at the test region in an amount in the range from about 0.1 mg/mL to about 10 mg/mL, preferably from about 0.2 mg/mL to about 10 mg/mL, preferably from about 0.3 mg/mL to about 10 mg/mL, preferably from about 0.4 mg/mL to about 10 mg/mL, preferably from about 0.5 mg/mL to about 10 mg/mL, preferably from about 0.6 mg/mL to about 10 mg/mL, preferably from about 0.7 mg/mL to about 10 mg/mL, preferably from about 0.8 mg/mL to about 10 mg/mL, preferably from about 0.9 mg/mL to about 10 mg/mL, preferably from about 1 mg/mL to about 10 mg/mL, preferably from about 2 mg/mL to about 10 mg/mL, or preferably from about 2 mg/mL to about 10 mg/mL In an embodiment, the capture antigen is a SARS-CoV-2 viral protein, or a functional variant thereof, as described herein, immobilised at the test region in an amount of at least about 0.1 mg/mL (i.e., 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 mg/mL, and so on), preferably at least about 0.1 mg/mL, preferably at least about 0.5 mg/mL, preferably at least about 1.0 mg/mL, preferably at least about 1.5 mg/mL, preferably at least about 2.0 mg/mL, preferably at least about 2.5 mg/mL, preferably at least about 3.0 mg/mL, preferably at least about 3.5 mg/mL, preferably at least about 4.0 mg/mL, preferably at least about 4.5 mg/mL, preferably at least about 5.0 mg/mL, preferably at least about 5.5 mg/mL, preferably at least about 6.0 mg/mL, preferably at least about 6.5 mg/mL, preferably at least about 7.0 mg/mL, preferably at least about 7.5 mg/mL, preferably at least about 8.0 mg/mL, preferably at least about 8.5 mg/mL, preferably at least about 9.0 mg/mL, preferably at least about 9.5 mg/mL, or more preferably at least about 10 mg/mL. In an embodiment, the immobilised capture antigen is a SARS- CoV-2 RBD, or a functional variant thereof, as described herein.

[0107] In an embodiment, the capture antigen is an ACE2 or an RBD-binding variant thereof (i.e., a SARS-CoV-2 RBD-binding variant thereof), as described herein, immobilised at the test region in an amount in the range from about 0.1 mg/mL to about 10 mg/mL, preferably from about 0.2 mg/mL to about 10 mg/mL, preferably from about 0.3 mg/mL to about 10 mg/mL, preferably from about 0.4 mg/mL to about 10 mg/mL, preferably from about 0.5 mg/mL to about 10 mg/mL, preferably from about 0.6 mg/mL to about 10 mg/mL, preferably from about 0.7 mg/mL to about 10 mg/mL, preferably from about 0.8 mg/mL to about 10 mg/mL, preferably from about 0.9 mg/mL to about 10 mg/mL, preferably from about 1 mg/mL to about 10 mg/mL, preferably from about 2 mg/mL to about 10 mg/mL, or preferably from about 2 mg/mL to about 10 mg/mL. In an embodiment, the capture antigen is an ACE2 or an RBD-binding variant thereof, as described herein, immobilised at the test region in an amount of at least about 0.1 mg/mL (i.e., 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 mg/mL, and so on), preferably at least about 0. 1 mg/mL, preferably at least about 0.5 mg/mL, preferably at least about 1.0 mg/mL, preferably at least about 1.5 mg/mL, preferably at least about 2.0 mg/mL, preferably at least about 2.5 mg/mL, preferably at least about 3.0 mg/mL, preferably at least about 3.5 mg/mL, preferably at least about 4.0 mg/mL, preferably at least about 4.5 mg/mL, preferably at least about 5.0 mg/mL, preferably at least about 5.5 mg/mL, preferably at least about 6.0 mg/mL, preferably at least about 6.5 mg/mL, preferably at least about 7.0 mg/mL,. preferably at least about 7.5 mg/mL, preferably at least about 8.0 mg/mL, preferably at least about 8.5 mg/mL, preferably at least about 9.0 mg/mL, preferably at least about 9.5 mg/mL, or more preferably at least about 10 mg/mL.

[0108] As noted elsewhere herein, the present inventors found that ACE2 does not bind very well to the LFA strip material, or that during use, the ACE2 may disassociates from the LFA strip material, resulting in an imprecise test result that could be construed as a false positive test result. The present inventors have unexpectedly found that ACE2 can be conjugated to a suitable carrier to assist in retaining the immobilised ACE2 to the LFA strip material during use such that there is no or minimal dissociation of the ACE2 from the test material during use. In an embodiment, the carrier is an Fc domain of an immunoglobulin molecule, or an Fc receptor (FcRN)-binding fragment thereof. Thus, in an embodiment disclosed herein, the capture antigen is an ACE2-Fc fusion protein. Suitable methods of producing an ACE2-Fc fusion protein will be familiar to persons skilled in the art, illustrative examples of which are described in Czajkowsky etal. (2012, EMBO Mol. Med , 4(10):0015-1028)), Yang et al. (2107; Front. Immunol:, 8: 1860) and Liu et al. (2018, Kidney Int:, 94(1): 114-125), the entire contents of which are incorporated herein by reference in their entirety.

[0109] The immobilised binding reagents disclosed herein (e.g., the capture antigens at the test region and /or the binding agents at the control region(s)) may suitably be impregnated throughout the thickness of the substrate as a bibulous or non-bibulous material in their respective regions. Such impregnation may enhance the extent to which the immobilized binding reagent(s) can bind to its binding partner during use. Alternatively, the immobilised binding reagents disclosed herein may be applied to the surface of the bibulous or non- bibulous material. Impregnation of specific binding members into test strip materials or application of specific binding members onto test strip materials may be done manually or by machine.

[0110] Nitrocellulose has the advantage that a specific binding reagent in the test and control regions can be immobilized without prior chemical treatment. If the porous solid phase material comprises paper, for example, the immobilization of the binding reagent in the test region can be performed by chemical coupling using, for example, CNBr, carbonyldiimidazole, ortresyl chloride.

[0111] Following the application of a specific binding reagent to the test and control regions, the remainder of the LFA material may suitably be treated to block any remaining binding sites elsewhere. Blocking can be achieved by any means known to persons skilled in the art, including by treatment with protein (e.g., bovine serum albumin or milk protein), or with polyvinylalcohol or ethanolamine, or by a combination of any of the foregoing.

[0112] Methods of applying or immobilising reagents to the LFA device will be familiar to persons skilled in the art. Various "printing" techniques have previously been used or known in the art for application of liquid reagents to carriers, for example micro-syringes, pens using metered pumps, direct printing and ink-jet printing, and any of these techniques can be used in the present context. To facilitate manufacture, the carrier (e.g., sheet) can be treated with the reagents and then subdivided into one or more of smaller portions, layers, components, laminates, or other structures (e.g., small narrow strips each embodying the required reagent-containing zones) to provide a plurality of identical carrier units.

[0113] The present disclosure also extends to automated, portable, and / or wireless LFA platforms for optical analysis of the results. For example, the platform may comprise digital image recognition software algorithms for qualitative and/or quantitative data test analysis and result reporting in a customizable software suite, with integrated alternative test strip/casing positioning for calibrated/result markings for digital image analysis to provide the test results in real time. The platform may optionally include digital camera hardware with digital components that record the results (detectable signals), software for interfacing with the user, and an image processing and computing device to interface with the digital camera. Digital image data of the test strip result, additional identifying information including one or more of identification of the person being tested, information about the tester, the location, the analyte(s) of interest, and the like, may be collected and stored in the digital camera, data memory storage, and/or a cloud based or separate data memory storage device. A digital image data is then processed using a host device (e.g. dedicated smart phone, PDA, laptop, cellular phone, or the like) using processing capabilities in conjunction with the software component of the system. Software pre-loaded onto the smart phone or processor provides the processing instructions and compares image analysis data to predefined calibration data, yielding a qualitative or quantitative result, e.g., but not limited to positive, negative, over or below one or more threshold concentrations or amounts, and the like.

[0114] In an embodiment, software is automated for LFA strip digital imaging for crossfield testing compatibility, which may suitably provide compatibility with a wide array of commercial or custom lateral flow strips. A system digitizes and objectively quantifies results from tests (such as test strips that can optionally be conventionally read by a human manually); stores original and modified digital image and data into memory for review; and enhances test processing by executing image processing algorithms.

[0115] In the context of LFA devices, the term "detecting" refers to any quantitative, semi- quantitative, or qualitative method, as well as to all other methods for determining the presence or concentration of analyte(s) of interest in general, and anti-viral antibodies in particular. For example, a method that merely detects the presence or absence of an antipsychotic drug in a sample lies within the scope of the present disclosure, as do methods that provide data as to the amount or concentration of the analyte(s) of interest in the sample. The terms "detecting", "determining", "identifying", and the like are used synonymously herein, and all lie within the scope of the present disclosure.

[0116] The various regions of the LFA device, including a sample application region(s), test region(s), and control region(s) can be on a single strip of material, such as fdter paper or nitrocellulose, or they can be provided on separate pieces of material. The different regions can be made of the same or different material or a combination of materials, but preferably are selected from bibulous materials, such as fdter paper, fiberglass mesh and nitrocellulose. [0117] In an embodiment, the regions of the LFA device are arranged in the following order, in the direction of flow: sample application region, conjugation region, test region, one or more control regions and, optionally, a fluid absorbing region. These regions can be provided in a single strip of a single material. Alternatively, the regions can be made of different materials and linked together in fluid communication. For example, the different regions can be in direct or indirect fluid communication. In this instance, the different regions can be jointed end-to-end to be in fluid communication, overlapped to be in fluid communication, or be communicated by another member, such an adjoining material, which is preferably bibulous such as fdter paper, fiberglass or nitrocellulose.

[0118] As noted elsewhere herein, the present disclosure provides a lateral flow assay that is capable of detecting viral infections. Instead of testing for analytes specific to a particular viral pathogen or viral infection, the LFA described herein tests for diagnostic markers of viral infection, more specifically markers produced in a host in response (e.g., following an immune response) to viral infection. The diagnostic markers (anti-viral antibodies) are preferably neutralising anti-viral antibodies; that is, antibodies capable of inhibiting entry of the virus into a cell of a host.

[0119] The LFA formats and devices described herein conveniently allow point of care diagnostic testing of viral infection (past or current infection). The LFA formats and devices described herein also conveniently allow point of care diagnostic testing to identify patients who are likely to have developed some degree of protective immunity against viral infection, including following immunisation with a vaccine designed to raise neutralising anti-viral antibodies in the patient. Such point of care diagnostic testing can be conveniently used, for example, in an outpatient clinical or during an urgent care visit, which can dramatically reduce health care costs by limiting misdiagnosis. The LFA formats and devices described herein also advantageously allow patients identified as having no or insufficient levels of neutralising anti-viral antibodies to be stratified to receive appropriate care, such as immunisation with a vaccine aimed at raising protective immunity (e.g., neutralising antibodies) against viral infection. Conversely, where the patient is identified as having a sufficient level of neutralising anti-viral antibodies as determined by the LFA devices and methods described herein, the cost and inconvenience of therapeutic treatment can be avoided. The relatively rapid result that can be obtained from the LFA-based test described herein also permits a diagnosis while the patient is still being examined by a medical practitioner. This, in another aspect, the present disclosure extends to a method of monitoring a subject for a viral infection or an antibody response against a viral infection, the method comprising using the LFA device as described herein to detect anti-viral antibodies in two or more biological fluid samples obtained from the subject at consecutive time points and making a determination as to whether the subject has developed an antibody response to the virus based on the change in the level of anti-viral antibodies in the two or more biological fluid samples obtained from the subject at the consecutive time points.

[0120] In an embodiment, the consecutive time points are at least 1 day apart, preferably at least 1 day apart, preferably at least 2 days apart, preferably at least 3 days apart, preferably at least 4 days apart, preferably at least 5 days apart, preferably at least 6 days apart, preferably at least 7 days apart, preferably at least 8 days apart, preferably at least 9 days apart, preferably at least 10 days apart, preferably at least 11 days apart, preferably at least 12 days apart, preferably at least 13 days apart, or more preferably at least 14 days apart.

[0121] In an embodiment, the LFA device comprises a chromatographic test strip with a test region and a control region, wherein the test and control regions are applied to the test strip in a linear (line) configuration. The fluid sample to be tested is applied to the sample application region of the chromatographic test strip. The sample then passes the conjugation region containing the labelled tracer antigen that is eluted by and then able to migrate with a sample transport liquid (e.g. a buffer solution) towards the test and control regions. The labelled tracer antigen may comprise a viral antigen that is capable of specifically binding to the anti-viral antibodies of interest when present in the sample to form a complex, thereby reducing the amount of free tracer antigen that can specifically bind to the capture antigen that is immobilised at the test region. Alternatively, the labelled tracer antigen may comprise a binding moiety capable of specifically binding to the viral antigen, such as a virus receptor or ligand binding variant thereof, which will compete for binding to the capture antigen (viral antigen) that is immobilised at the test region with anti-viral antibodies that may be present in the sample. An absorbent pad, as well as other known LFA components such as a waste zone, a carrier backing, a housing, and an opening in the housing for result read out, may optionally also be a component of the LFA device.

[0122] At the test region, the presence of the anti-viral antibodies from the sample is determined by a qualitative and/or quantitative readout of the test region indication resulting from the accumulation of labelled tracer antigen. The test strip may further comprise one or more additional test regions to detect other viral and/or viral infection markers, as described elsewhere herein.

[0123] The control region indicates that the labelled tracer antigen has flowed through the length of the test strip, even though the device has provided a negative result (i.e., absence of the anti-viral antigens in the sample), thus confirming proper operation of the assay. The control region is suitably downstream of the test region(s). However, in some embodiments, the control region may be located upstream of any one or more of the test regions. In an embodiment, the control region comprises an antibody or other binding moiety that binds to a component of the elution medium or other composition being used in the test. In some embodiments, the control region comprises an antibody or other binding moiety that binds specifically to the detectable moiety or label of the tracer antigen.

Viral antigens, viral receptors and functional variants thereof

[0124] It will be understood that the viral antigen or functional variant thereof will depend on the specificity of the anti-viral antibodies the LFA device used to detect. For example, where the LFA device is to be used to detect anti-influenza A antibodies in a sample, the viral antigen will suitably be an antigen derived from a strain of influenza A. Likewise, where the LFA device is to be used to detect anti-SARS-CoV-2 antibodies in a sample, the viral antigen will suitably be an antigen derived from SARS-CoV-2. Thus, advantageously, the LFA device disclosed herein is not limited to the detection of antibodies that bind to specific viral antigens and can suitably be adapted to the detection of antibodies, including neutralising antibodies, which bind to any viral antigen of interest. [0125] In some embodiments, the viral antigen is isolated from a virus that is propagated in culture, methods of which will be familiar to persons skilled in the art. In an embodiment, the viral antigen comprises, consists or consists essentially of a native or naturally-occurring viral antigen. In an embodiment, the viral antigen is non-naturally occurring; for example, the viral antigen may suitably be synthesised, including by recombinant technology, methods of which will be familiar to persons skilled in the art. In an embodiment, the non- naturally occurring viral antigen will suitably have an amino acid sequence that shares 100% sequence identity or sequence homology with the amino acid sequence of the native viral antigen. In other embodiments, the non-naturally occurring viral antigen may have an amino acid sequence that shares less than 100% sequence identity or similarity with a native viral antigen, but is still capable of binding to anti-viral antibodies in a biological sample that were generated or raised against the native virus (e.g., following infection by the virus) or against the viral antigen (e.g., following immunisation). The viral antigen may differ from the native viral antigen sequence by one or more amino acid insertions, deletions or substitutions and/or by one or more other modifications. The present disclosure thus extends to functional variants of native viral antigens.

[0126] A “functional variant”, as used herein, means a peptide sequence that has a different amino acid sequence to a peptide sequence to which it is being compared (z. e. , a comparator), including a native or naturally-occurring peptide sequence, yet retains the ability to bind to anti-viral antibodies in the sample, including anti-viral antibodies that have been raised against the native virus or viral antigen. Functional variants, as used herein, extend to fragments of native sequences, yet retains the ability to bind to anti-viral antibodies in the sample, including anti-viral antibodies that have been raised against the native virus or viral antigen.

[0127] Suitable methods of determining whether a functional variant retains the ability to bind to anti-viral antibodies in the sample, including anti-viral antibodies that have been raised against the native virus or viral antigen, will be familiar to persons skilled in the art, illustrative examples of which include western blot and enzyme-linked immunosorbent assays. The functional variant will suitably be compared to the native viral antigen for its ability to bind to an anti-viral antibody. In an embodiment, the functional variant comprises, consists, or consists essentially of an amino acid sequence that differs from the native peptide sequence by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) amino acid substitutions, wherein said difference does not, or does not completely, abolish the ability of the variant to bind to an anti-viral antibody, including an anti-viral antibody raised against the native virus or viral antigen. In an embodiment, the functional variant differs from the native sequence by one or more conservative amino acid substitutions. As used herein, the term “conservative amino acid substitution” refers to changing amino acid identity at a given position to replace it with an amino acid of approximately equivalent size, charge and/or polarity. Examples of natural conservative substitutions of amino acids include the following 8 substitution groups (designated by the conventional one-letter code): (1) M, I, L, V; (2) F, Y, W; (3) K, R, (4) A, G; (5) S, T; (6) Q, N; (7) E, D; and (8) C, S.

[0128] In an embodiment, the viral antigen has an amino acid sequence that shares at least 70% sequence identity or similarity to the amino acid sequence of the native viral antigen. Reference to "at least 70%" includes 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or similarity, for example, after optimal alignment or best fit analysis. In an embodiment, the viral antigen has at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% or preferably 100% sequence identity or similarity to the amino acid sequence of the native viral antigen after optimal alignment or best fit analysis.

[0129] The terms “identity”, “similarity”, “sequence identity”, “sequence similarity”, “homology”, “sequence homology” and the like, as used herein, mean that at any particular amino acid residue position in an aligned sequence, the amino acid residue is identical between the aligned sequences. The term “similarity” or “sequence similarity” as used herein, indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, leucine may be substituted for an isoleucine or valine residue. This may be referred to as conservative substitution. In an embodiment, the amino acid sequences may be modified by way of conservative substitution of any of the amino acid residues contained therein, such that the modification has no effect on the binding specificity or functional activity of the modified polypeptide when compared to the unmodified polypeptide. [0130] In some embodiments, sequence identity with respect to an amino acid sequence relates to the percentage of amino acid residues in the candidate sequence which are identical with the residues of the corresponding peptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C- terminal extensions, nor insertions shall be construed as reducing sequence identity or homology. Methods and computer programs for performing an alignment of two or more amino acid sequences and determining their sequence identity or homology are well known to persons skilled in the art. For example, the percentage of identity or similarity of two amino acid sequences can be readily calculated using algorithms, for example, BLAST, FASTA, or the Smith-Waterman algorithm.

[0131] Techniques for determining an amino acid sequence "similarity" are well known to persons skilled in the art. In general, "similarity" means an exact amino acid to amino acid comparison of two or more amino acid sequences or at the appropriate place, where amino acids are identical or possess similar chemical and/or physical properties such as charge or hydrophobicity. A so-termed "percent similarity" then can be determined between the compared peptide sequences. In general, "identity" refers to an exact amino acid to amino acid correspondence of two amino acid sequences.

[0132] Two or more amino acid sequences can also be compared by determining their "percent identity". The percent identity of two sequences may be described as the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be extended to use with peptide sequences using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). Suitable programs for calculating the percent identity or similarity between sequences are generally known in the art.

[0133] Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res.25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel etal., "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15.

[0134] As noted elsewhere herein, the choice of viral antigen or functional variant thereof may depend on the type of anti-viral antibodies the LFA device is used to detect and can therefore be derived from any virus. Typically, the viral antigen will be of a virus that is known to cause infection. In an embodiment, the viral antigen will be a ligand for which the receptor that facilitates viral entry into a cell (i.e., infection) is known.

[0135] In an embodiment, the viral antigen is of a virus selected from the group consisting of a picomavirus, a coronavirus, an influenza virus, a parainfluenza virus, a respiratory syncytial virus, an adenovirus, an enterovirus, and a metapneumo virus. Suitable antigens of picomavirus, a coronavirus, an influenza vims, a parainfluenza vims, a respiratory syncytial vims, an adenovims, an enterovims, and a metapneumovims will be familiar to persons skilled in the art, illustrative examples of which are described elsewhere herein.

[0136] In an embodiment, the vims is of the family Coronaviridae (see Payne, S: Chapter 17 - Family Coronaviridae; Viruses: From Understanding to Investigation, 2017, Pages 149-158; and Family - Coronaviridae: Vims Taxonomy, Ninth Report of the International Committee on Taxonomy of Vimses, 2012, Pages 806-828). The Coronaviridae family is typically divided into Coronavirinae and Torovirinae sub-families, which are further divided into six genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, Deltacoronavirus, Torovirus, and Bafinivirus. While vimses in the genera Alphacoronaviruses and Betacoronaviruses infect mostly mammals, the Gammacoronavirus infect avian species and members of the Deltacoronavirus genus have been found in both mammalian and avian hosts (see, e.g., Phan etal., Virus Evol. 2018; 4(2): vey035). Coronavimses (CoV) cause a range of respiratory, enteric, and neurological diseases in human and animals. In human CoV infections, the severe acute respiratory syndrome coronavims (SARS-CoV) and Middle East respiratory syndrome coronavims (MERS-CoV) cause severe respiratory tract disease with high mortality rates, and there is strong evidence of zoonosis for both vimses. Given the zoonotic movement, detailed descriptions of the Coronaviridae in broad animal reservoirs that may cross the host barriers to cause diseases in humans are important. All Coronaviridae family members typically share the same unique strategy for mRNA synthesis whereby the polymerase complex jumps or moves from one region of the template to a more distant region. The need for the polymerase complex to dissociate from the template may explain the high rate of RNA recombination that occurs during genome replication.

[0137] Suitable viruses of the family Coronaviridae will be familiar to persons skilled in the art, illustrative examples of which include Alphaletovirus (see, e.g, Bukhari etal.,' Virology. 2018; 524: 160-171) and Coronavirus (see, e.g., Fehr and Perlman; Coronaviruses. 2015; 1282: 1-23). Thus, in an embodiment disclosed herein, the virus is selected from the group consisting of Alphaletovirus and Coronavirus. In an embodiment, the virus is a coronavirus. In an embodiment disclosed herein, the coronavirus is selected from the group consisting of Alphacoronavirus, Betacoronavirus, Deltacoronavirus and Gammacoronavirus. In an embodiment, the coronavirus is Betacoronavirus. Suitable Betacoronaviruses will be familiar to persons skilled in the art, an illustrative example of which includes a Sarbecovirus. In an embodiment, the Betacoronavirus is a Sarbecovirus. Suitable Sarbecoviruses will be familiar to persons skilled in the art, illustrative examples of which include Severe acute respiratory syndrome-related coronavirus, Severe acute respiratory syndrome coronavirus (SARS-CoV; see, e.g., Vijayanand et al., Clin Med (Lond). 2004; 4(2): 152-60) and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; see, e.g., Khailany et al. Gene Rep. 2020; 19: 100682). In an embodiment, the Sarbecovirus is selected from the group consisting of Severe acute respiratory syndrome-related coronavirus, SARS-CoV and SARS-CoV-2. In an embodiment, the Sarbecovirus is SARS- CoV-2. In an embodiment, the SARS-CoV-2 is encoded by a nucleic acid sequence of NCBI Accession Number NC_045512. In an embodiment, the virus is a SARS-CoV-2 virus. Examples of SARS-CoV-2 viruses will be familiar to persons skilled in the art, illustrative examples of which are included in the GISAID database (https : //ww . gi said . org) . such as the Wuhan-Hu-1 virus (GenBank accession no. NC_045512; European Nucleotide Archine reference no. MN908947.3; also known as the Wuhan-Hu-1 reference genome) and variants thereof, such as the S477N variant, the S477I variant and the N439K variant, where S477N, S477I and N439K refer to amino acid substitutions at positions corresponding to the reference Wuhan Hu-1 RBD sequence shown in NC_045512. The Wuhan virus strains have also been recorded as Wuhan/IVDC-HB-01/2019 (GISAID accession ID:EPI_ISL_402119) (HB01), Wuhan/IVDCHB-04/2019 (EPI ISL 402120) (HB04), and Wuhan/IVDC-HB- 05/2019 (EPI_ ISL_402121) (HB05). Other variants of SARS-CoV-2 have been identified and vary from the Wuhan-Hu-1 reference sequence by amino acid substitutions N501Y, E484K, K417N, K417T and combinations thereof, examples of which include the UK (B. 1.1.7), the South African (B.1.351) and the Brazillian strains (P. l)). In an embodiment, the viral antigen comprises an amino acid sequence of the RBD antigen, or an ACE2 -binding fragment thereof, derived from the Wuhan-Hu-1 reference sequence (NC_045512), or a variant comprising an amino acid substitution selected from the group consisting of S477N, S477I, N439K, N501Y, E484K, K417N, and K417T and any combination of the foregoing (when compared to the Wuhan-Hu- 1 RBD reference sequence). In an embodiment, the viral antigen comprises, consists, or consists essentially of, an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, or more preferably at least 99% sequence identity to SEQ ID NO: 1.

SEQ IDNO: 1 (RBD protein sequence - residues R319 to F541 ofthe SARS-CoV-2 S protein described in UniProtKB - PODTC2):

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFN CYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0138] As noted elsewhere herein, the present disclosure extends to functional variants, including of viral antigens, insofar as such variants have a peptide sequence that has a different amino acid sequence to a peptide sequence to which it is being compared (i.e., a comparator), including a native or naturally-occurring peptide sequence, yet retains the ability to bind to anti-viral antibodies in the sample, including anti-viral antibodies that have been raised against the native virus or viral antigen. Functional variants, as used herein, extend to fragments of native sequences, yet retains the ability to bind to anti-viral antibodies in the sample, including anti-viral antibodies that have been raised against the native virus or viral antigen. [0139] In an embodiment, the functional variant is a fragment of the viral antigen. In a further embodiment, the functional variant is a fragment of the viral antigen SARS-CoV-2 RBD. Methods for identifying whether a fragment of a viral antigen is a functional variant, insofar as it retains the ability to bind to anti-viral antibodies in a sample, including antiviral antibodies that have been raised against the native virus or viral antigen, would be familiar to persons skilled in the art, illustrative examples of which are described herein. In an embodiment, the viral antigen comprises, consists or consists essentially of an amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, or more preferably at least 99% sequence identity to SEQ ID NO:2.

SEQ ID NO:2 (an N- and C-terminal truncated RBD protein sequence - residues N334 to P527 of the SARS-CoV-2 S protein described in UniProtKB - PODTC2):

NLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQP TNGVGYQPYRVVVLSFELLHAPATVCGP

[0140] In another embodiment, the viral antigen comprises, consists or consists essentially of an amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, or more preferably at least 99% sequence identity to SEQ ID NO:3.

SEQ ID NO:3 (an N- and C-terminal truncated RBD protein sequence - residues 1332 to N532 of the SARS-CoV-2 S protein described in UniProtKB - PODTC2):

ITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKL NDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLD SKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQ PTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTN

[0141] In an embodiment, the SARS-CoV-2 vims is selected from the group consisting of the Wuhan-Hu- 1 vims strain, the UK vims strain (B. 1.1.7), the South African vims strain (B. 1.351) and the Brazillian vims strain (P.1)). Persons skilled in the art will understand that the devices and methods disclosed herein are applicable to the detection of neutralising antiviral antibodies against any viral antigen, including new and emerging variants.

[0142] In an embodiment, the viral antigen comprises, consists, or consists essentially of a SARS-CoV-2 spike protein, the amino acid sequence of which will be familiar to persons skilled in the art and otherwise described in the literature. In an embodiment, the SARS- CoV-2 spike protein comprises, consists, or consists essentially of an amino acid sequence that has at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% or preferably 100% sequence identity to an amino acid sequence encoded by the nucleic acid sequence disclosed in GenlD 43740568. In an embodiment, the SARS-CoV-2 spike protein comprises, consists, or consists essentially of an amino acid sequence that has at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% or preferably 100% sequence identity to the amino acid sequence disclosed in NCBI Accession No. YP_009724390.

[0143] In an embodiment, the viral antigen comprises, consists, or consists essentially of a receptor binding domain (RBD) of the SARS-CoV-2 spike protein. In an embodiment, the RBD of the SARS-CoV-2 spike protein comprises, consists, or consists essentially of an amino acid sequence that has at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% or preferably 100% sequence identity to an amino acid sequence comprising amino acid residues R319 to F541 of SARS-CoV-2 as described, for example, in Lan et al. (2020; Nature,' 581:215-220), the contents of which is incorporated herein by reference in its entirety.

[0144] As noted elsewhere herein, the type of virus receptor will depend on the type of antiviral antibody for which the LFA device is to be used to detect. For example, where the LFA device is to be used to detect anti-influenza A antibodies in a sample, the virus receptor may be haemagglutinin (HA). Likewise, where the LFA device is to be used to detect anti- SARS-CoV-2 antibodies in a sample, the virus receptor may be Angiotensin Converting Enzyme 2 (ACE2).

[0145] In an embodiment, the virus receptor is isolated from a cell that is propagated in culture, methods of which will be familiar to persons skilled in the art. Such virus receptors may be referred to herein as native or naturally-occurring virus receptors. In some embodiments, the virus receptor is non-naturally occurring; that is, the virus receptor may suitably be synthesised, including by recombinant technology, methods of which will also be familiar to persons skilled in the art. In an embodiment, the non-naturally occurring virus receptor will suitably have an amino acid sequence that shares 100% sequence identity or sequence homology with the amino acid sequence of the native virus receptor. In other embodiments, the non-naturally occurring viral antigen may have an amino acid sequence that shares less than 100% sequence identity or similarity with a native virus receptor, but is still capable of binding to the viral antigen. The viral antigen may differ from the native virus receptor sequence by one or more amino acid insertions, deletions or substitutions and/or by one or more other modifications. The present disclosure therefore extends to functional variants of native virus receptors. It is to be understood that a “functional variant”, in this context, means a peptide sequence that has a different amino acid sequence to a native virus receptor to which it is being compared (i.e., a comparator), yet retains the ability to bind to the viral antigen or to a functional variant thereof that is to be employed in the LFA device. Functional variants, as used herein, extend to fragments of the native sequence of the virus receptor to which it is being compared, yet retains the ability to bind to the viral antigen or functional variant thereof employed in the LFA device. Suitable variants will be familiar to persons skilled in the art. For example, where the LFA device is employed to detect anti-SARS-CoV-2 antibodies in a sample, the functional variant of the vims receptor is a receptor binding domain (RBD)-binding fragment of ACE2.

[0146] Suitable methods of determining whether a functional variant of the vims receptor retains the ability to bind to the viral antigen or functional variant thereof will be familiar to persons skilled in the art, illustrative examples of which include western blot and enzyme- linked immunosorbent assays, where the variant can be compared to the native sequence for its ability to bind to the viral antigen or functional variant thereof.

[0147] As noted elsewhere herein, a functional variant may include an amino acid sequence that differs from the native peptide sequence by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) amino acid substitutions, wherein said difference does not, or does not completely, abolish the ability of the variant to bind to the viral antigen. In an embodiment, the functional variant differs from the native sequence by one or more conservative amino acid substitutions. As used herein, the term “conservative amino acid substitution” refers to changing amino acid identity at a given position to replace it with an amino acid of approximately equivalent size, charge and/or polarity. Examples of natural conservative substitutions of amino acids include the following 8 substitution groups (designated by the conventional one-letter code): (1) M, I, L, V; (2) F, Y, W; (3) K, R, (4) A, G; (5) S, T; (6) Q, N; (7) E, D; and (8) C, S.

[0148] In an embodiment, the virus receptor has an amino acid sequence that shares at least 70% sequence identity or similarity to the amino acid sequence of the native virus receptor. Reference to "at least 70%" includes 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or similarity, for example, after optimal alignment or best fit analysis. In an embodiment, the virus receptor has at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% or preferably 100% sequence identity or similarity to the amino acid sequence of the native virus receptor after optimal alignment or best fit analysis.

[0149] As noted elsewhere herein, the choice of virus receptor will depend on the type of anti-viral antibodies the LFA device is employed to detect. It is therefore to be understood that the amino acid sequence of the vims receptor can suitably be derived from any vims. Typically, the vims receptor will have been identified as a receptor that facilitates viral entry into a cell and subsequent infection. In an embodiment, the vims receptor selected from the group consisting of a picomavims, a coronavims, an influenza vims, a parainfluenza vims, a respiratory syncytial vims, an adenovims, an enterovims, and a metapneumovims, illustrative examples of which are described elsewhere herein.

[0150] In an embodiment, the vims receptor is a receptor that facilitates entry into a cell of a vims of the family Coronaviridcie. As noted elsewhere herein, suitable vimses of the family Coronaviridae will be familiar to persons skilled in the art, illustrative examples of which include Alphaletovims (see, e.g., Bukhari et al , Virology. 2018; 524: 160-171) and Coronavims (see, e.g., Fehr and Perlman; Coronaviruses . 2015; 1282: 1-23). Thus, in an embodiment disclosed herein, the vims receptor is a receptor that facilitates entry into a cell of a vims selected from the group consisting of Alphaletovims and Coronavims. In an embodiment, the vims receptor is a receptor that facilitates entry into a cell of a coronavims. In an embodiment, the coronavims is selected from the group consisting of Alphacoronavims, Betacoronavims, Deltacoronavims and Gammacoronavims. In an embodiment, the coronavims is Betacoronavims. Suitable Betacoronavimses will be familiar to persons skilled in the art, an illustrative example of which includes a Sarbecovims. Thus, in an embodiment, the Betacoronavims is a Sarbecovims.

[0151] Suitable Sarbecovimses will be familiar to persons skilled in the art, illustrative examples of which include Severe acute respiratory syndrome-related coronavims, Severe acute respiratory syndrome coronavims (SARS-CoV; see, e.g., Vijayanand et al., Clin Med (Lond). 2004; 4(2): 152-60) and Severe acute respiratory syndrome coronavims 2 (SARS- CoV-2; see, e.g., Khailany et al. Gene Rep. 2020; 19: 100682). In an embodiment disclosed herein, the Sarbecovims is selected from the group consisting of Severe acute respiratory syndrome-related coronavims, SARS-CoV and SARS-CoV-2. In an embodiment, the Sarbecovims is SARS-CoV-2. In an embodiment, the SARS-CoV-2 is encoded by a nucleic acid sequence of NCBI Accession Number NC_045512.

[0152] In an embodiment, the vims receptor is a receptor that facilitates entry of SARS- CoV-2 into a cell. In an embodiment, the vims receptor is ACE2. In an embodiment, the vims receptor comprises, consists or consists essentially of an amino acid sequence that has at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% or preferably 100% sequence identity to the amino acid sequence disclosed in GenBank Accession Number NM_001371415 or GenBank Accession Number NM_001358344.

[0153] In an embodiment, the functional variant of the virus receptor comprises, consists, or consists essentially of an RBD-binding fragment of ACE2; that is, a fragment of ACE2 that is capable of binding to the receptor binding domain (RBD) of SARS-CoV-2, as described elsewhere herein.

[0154] The present disclosure also extends to non-human variants (isoforms) of virus receptors, illustrative examples of which will be familiar to persons skilled in the art and include canine, feline, equine and porcine isoforms. For instance, canine ACE2 includes the isoform described in GenBank Accession Number NM_001158732; feline ACE2 includes the isoform described in GenBank Accession Number NM_001034565; equine ACE2 includes the isoform described in GenBank Accession Number XP_001490241 and porcine ACE2 includes the isoform described in GenBank Accession Number NM_001116542.

Control region and immobilised binding agents

[0155] The LFA device may suitably comprises a control region for the purpose of detecting proper operation of the device, including proper flow of the analyte(s) of interest from the sample application and conjugation regions, in the direction of flow.

[0156] Thus, in an embodiment, the lateral flow assay device described herein further comprises a control region comprising an immobilised control agent. As used herein, the term "immobilised control agent" means a molecule that is capable of forming a complex with a component present in the fluid sample, elution buffer and / or with the tracer antigen, wherein formation of said complex provides a detectable control signal at the control region. It will therefore be understood that the type of immobilised control agent is not limiting to the application of this embodiment of the LFA device described herein, as long as it is capable of forming a detectable signal at the control region in the presence of a component in the fluid sample, elution buffer and / or with the tracer antigen. [0157] In an embodiment, the immobilised control agent forms a complex with a labelled component of the fluid sample or with the tracer antigen to produce a detectable control signal at the control region. In an embodiment, the immobilised control agent forms a complex with the tracer antigen to produce a detectable control signal at the control region. In an embodiment, the control signal is representative of a test signal that is produced at the test region in absence of the anti-viral antibody. This embodiment advantageously allows the test result to be determined by a comparison of the test signal to the control signal located on the same device, wherein a weaker test signal when compared to the control signal is indicative of the presence of anti-viral antibodies in the sample. Conversely, a test signal that is comparable (e.g., of equal or substantially equal intensity) to the control signal is indicative of the absence of anti-viral antibodies in the sample. Alternatively, or in addition, the control region may be located on a separate LFA device to the test region, which may be referred to as a control LFA device or control strip that can optionally be run in parallel with and compared to the test signal obtained at the test region. In some embodiments, the control and test LFA devices are configured in a single housing (e.g., a cassette), as described elsewhere herein. Suitable configurations will be familiar to persons skilled in the art and may suitably include side-by-side, end-to-end and back-to-back. In an embodiment, the control LFA device and the test LFA devices are configured side-by-side.

[0158] Where the control region is located on the LFA device with the test region, the control region can be upstream from, or downstream from, the test region. However, it is generally desirable to configure the LFA device so that the control region is downstream from the test region to ensure that a negative result at the test region is a true negative result and not a result of insufficient flow of analytes from the sample application and conjugation zones in the direction of flow. The control region therefore provides a result that indicates that the test has performed correctly. Thus, in an embodiment, the control region is downstream of the test region in the direction of flow.

[0159] The control region will suitably comprise an immobilised binding agent that is capable of binding to, and forming a complex with, the tracer antigen from the conjugation region so as to produce a detectable signal at the control region, also referred to herein as a detectable control signal.

[0160] The LFA device disclosed herein may optionally comprise more than one control region. For example, the LFA device may be configured to carry a second control region, which may be upstream from, or downstream from, the aforementioned first control region. However, it is generally desirable to configure the LFA device so that the two or more control regions are downstream of the test region in the direction of flow. In an embodiment, the second control region comprises immobilised second binding agents capable of binding specificity to immunoglobulin, including specific isotypes thereof (e.g., IgG, IgM), thereby providing an indication of the presence of total immunoglobulin or immunoglobulin isotypes in the sample. In some circumstances, this can be advantageous by providing a positive control for the presence of immunoglobulin in the sample, irrespective of whether the test region gives a positive or negative result. For example, where a positive result (i.e., a detectable signal) is obtained at the second control region and a negative result (i.e., a detectable signal) is obtained at the test region, this is indicative of the absence of anti-viral antibodies in the sample. A given LFA device may therefore comprise a single control region or two or more different control regions, where the immobilized control agents of each region may be the same or different. The control binding agent may optionally be non- stably associated with the bibulous member at a location that is upstream from the control region.

[0161] In an embodiment, the LFA device disclosed herein may comprise two or more test strips suitably configured to allow parallel strips (within parallel cassettes) running the same sample in parallel flow but detecting the presence of different neutralizing antibodies for the two or more strips/cassettes. For example, the first might detect Wuhan virus-neutralizing antibodies, and then in an attached and parallel cassette, the sample also flows along another strip configured with reagents to specifically detecting neutralizing antibodies of a different variant or strain of coronavirus, or even other viruses, etc. which may be referred to as a control LFA device or control strip that can optionally be run in parallel with and compared to the test signal obtained at the test region. In some embodiments, the control and test LFA devices are configured in a single housing (e.g., a cassette), as described elsewhere herein. Suitable configurations will be familiar to persons skilled in the art and may suitably include side-by-side, end-to-end and back-to-back. In an embodiment, the control LFA device and the test LFA devices are configured side-by-side.

[0162] The LFA device may optionally include an absorbent material (e.g., pad) downstream from the test and control regions, where the absorbent material is configured to absorb fluid and reagents present therein that have flowed along the device in the direction of flow.

[0163] In another aspect disclosed herein, there is provided a method of detecting an antiviral antibody in a biological fluid sample of a subject, the method comprising:

[0164] In an embodiment, the reference test signal is representative of a detectable test signal produced by a biological fluid sample that does not contain the anti-viral antibody.

[0165] In another aspect disclosed herein, there is provided a method of identifying a subject as being a source of neutralising anti-viral antibodies, the method comprising: a) obtaining a biological fluid sample from a subject; b) applying the biological fluid sample from step (a) to the sample application region of the lateral flow assay device, as described herein, for a period of time sufficient to allow the biological fluid sample and trace antigen to flow to the test region; and c) comparing the detectable test signal at the test region with a reference test signal, wherein the subject is identified as a source of neutralising anti-viral antibodies when a weaker test signal is detected at the test line when compared to the reference test signal. [0166] In an embodiment, the reference test signal is representative of a detectable test signal produced by a biological fluid sample that does not contain neutralising anti-viral antibodies. [0167] In an embodiment, the biological fluid is selected from the group consisting of blood, serum, plasma, saliva and nasopharyngeal secretion.

[0168] The present disclosure also extends to a composition enriched for neutralising antiviral antibodies obtained from the source identified by the method disclosed herein.

[0169] In another aspect disclosed herein, there is provided a method of treating or preventing viral infection in a subject in need thereof, the method comprising administering to the subject the composition described herein. [0170] The present disclosure also extends to a method of identifying the presence of a neutralising anti-viral antibody in a sample, the method comprising: a) applying a sample to the sample application region of the lateral flow assay device, as described herein, for a period of time sufficient to allow the sample and trace antigen to flow to the test region; and b) comparing the detectable test signal at the test region with a reference test signal, wherein the subject is identified as a source of neutralising anti-viral antibodies when a weaker test signal is detected at the test region when compared to the reference test signal. [0171] In an embodiment, the reference test signal is representative of a detectable test signal produced by sample that does not contain neutralising anti-viral antibodies.

[0172] In an embodiment, the reference signal is the control signal, as described elsewhere herein. This embodiment advantageously allows the test result to be determined by a comparison of the test signal to the control signal located on the same device, wherein a weaker test signal when compared to the control signal is indicative of the presence of antiviral antibodies in the sample. Conversely, a test signal that is comparable (e.g., of equal or substantially equal intensity) to the control signal is indicative of the absence of anti-viral antibodies in the sample. In this context, the control region is suitably calibrated so as to provide a detectable signal that is sufficiently representative of a test signal obtained at the test region in the absence of anti-viral antibodies, more particularly in the absence of neutralizing anti-viral antibodies. By "sufficiently representative" means that the detectable control signal is of equal or substantially equal intensity or value to a test signal obtained at the test region in the absence of neutralizing anti-viral antibodies. In an embodiment, the control region is suitably calibrated so as to provide a detectable control signal that is at least about 70%, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, preferably at least about 90%, preferably at least about 95%, or more preferably about 100% of the intensity or value of the detectable test signal obtained at the test region in the absence of anti-viral antibodies. In an embodiment, the control region is suitably calibrated so as to provide a detectable control signal that is within about 30%, preferably within about 25%, preferably within about 25%, preferably within about 20%, preferably within about 15%, preferably within about 10%, preferably within about 5%, or more preferably within about 2% of the intensity or value of the detectable test signal obtained at the test region in the absence of anti-viral antibodies. [0173] The control region may suitably be calibrated to provide a detectable signal that is sufficiently representative of a detectable test signal obtained from a sample comprising neutralizing anti-viral antibodies. Thus, in an embodiment, the detectable control signal is representative of a test signal that is produced at the test region in the presence of the antiviral antibody. In this context, the reference or control regions may aid in the quantitative or semi-quantitative estimation of the presence of neutralizing anti-viral antibodies and the % inhibition at the test region, without the need for performing additional controls in separate tests. The intensity of the reference or control signal may suitably be adjusted or calibrated during manufacture by altering the concentration of the immobilised capture antigen in the reference / control region to provide a detectable reference / control signal at the reference / control region that is independent of the test sample. The intensity of the detectable reference / control signal can be calibrated or adjusted so that it is representative of a desired level of inhibition at the test region (as indicative of the presence of neutralizing anti-viral antibodies), including when compared to a detectable test signal that is generated using a representative negative control sample (i.e., samples in which neutralizing anti-viral antibodies are absent or undetectable). For instance, the reference / control region may be set at 50% of the average intensity of test signals generated from representative samples of healthy subjects that are known not to comprise neutralising anti-viral antibodies, so that a test sample that gives the same or substantially the same detectable test signal at the test region is indicative that the test sample comprises an amount of neutralizing anti-viral antibodies that represent 50% inhibition; a detectable test signal that is stronger in intensity than the reference signal is indicative of less than 50% inhibition; a detectable test signal that is twice as strong as the reference signal may be indicative of 0% inhibition; a detectable test signal that has an intensity that is weaker than the reference signal is indicative of >50% inhibition; and the absence of a detectable test signal is indicative of 100% inhibition at the test region. In this context, the % inhibition that is detected at the test region can be used as a surrogate for the concentration of neutralizing anti-viral antibodies in the sample.

[0174] In an embodiment, the LFA device comprises a control region that is calibrated to provide a detectable control signal that has an intensity or value that is at least about 30%, preferably at least about 35%, preferably at least about 40%, preferably at least about 45%, preferably at least about 50%, preferably at least about 55%, preferably at least about 60%, preferably at least about 65%, preferably at least about 70%, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, preferably at least about 90%, preferably at least about 95%, or preferably about 100% of the intensity or value that is representative of a detectable test signal generated from a sample that does not comprise, or substantially comprise, neutralizing anti-viral antibodies. In an embodiment, the intensity or value of the detectable control signal is the average or mean of detectable test signals generated from two or more samples that do not comprise, or substantially comprise, neutralizing anti-viral antibodies (e.g., from healthy subjects who have not been prior exposed to the viral antigen in question). In an embodiment, the control region is calibrated to provide a detectable control signal that has an intensity or value that is from about 30% to about 70% of the intensity or value that is representative of a detectable test signal generated from a sample that does not comprise, or substantially comprise, neutralizing antiviral antibodies. Thus, in an embodiment, the control signal has an intensity or value of from about 30% to about 70% of the intensity or value that is representative of the test signal produced at the test region in absence of the anti-viral antibody. In another embodiment, the control region is calibrated to provide a detectable control signal that has an intensity or value that is from about 40% to about 60% of the intensity or value that is representative of a detectable test signal generated from a sample that does not comprise, or substantially comprise, neutralizing anti-viral antibodies. In an embodiment, the control region is calibrated to provide a detectable control signal that has an intensity or value that is about 50% of the intensity or value that is representative of a detectable test signal generated from a sample that does not comprise, or substantially comprise, neutralizing anti-viral antibodies. [0175] In an embodiment, LFA device is stable for at least about 1 month, 3 months, 6 months, 12 months, 18 months, or 24 months when stored at about 4°C to about 30°C. In an embodiment, the LFA device is stable for about 1 to about 6, about 1 to about 12, about 1 to about 18, or about 1 to about 24 months when stored at about 4°C to about 30°C.

[0176] In an embodiment, the LFA device is stable for at least about 1 month, 3 months, 6 months, 12 months, 18 months, or 24 months when stored at room temperature. In embodiments, a assay or device composition of the present invention is stable for about 1 to about 6, about 1 to about 12, about 1 to about 18, or about 1 to about 24 months when stored at room temperature.

[0177] By "stable" is meant that the ability of the LFA device to be used to detect the analyte(s) of interest (anti-viral antibodies) in a sample is not adversely affected. In some embodiments, the level of sensitivity and / or selectivity of the LFA device to detect the analyte(s) of interest will remain substantially unchanged during storage for at least about 1 month, 3 months, 6 months, 12 months, 18 months, or 24 months at room temperature or at about 4°C to about 30°C. By "substantially unchanged" means that the level of sensitivity and / or selectivity of the LFA device to detect the analyte(s) of interest in a sample will be at least about 50%, preferably at least about 55%, preferably at least about 60%, preferably at least about 65%, preferably at least about 70%, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, preferably at least about 90%, preferably at least about 95%, or more preferably 100% of the level of sensitivity and / or selectivity of the LFA device to detect the analyte(s) of interest at a time before storage,

[0178] Application of the sample to the sample application region of the LFA device can be carried out by use of any appropriate instrument as known in the art, e g., a dropper or pipet, to apply an appropriate sample volume.

[0179] In an embodiment, the LFA result is ready to read in about 5 to about 20 minutes. In an embodiment, the LFA result is ready to read in about 5, preferably about 6, preferably about 7, preferably about 8, preferably about 9, preferably about 10, preferably about 11, preferably about 12, preferably about 13, preferably about 14, preferably about 15 preferably about 16, preferably about 17, preferably about 18, preferably about 19 preferably about 20, preferably about 21, preferably about 22, preferably about 23 preferably about 24, preferably about 25, or more preferably about 30 minutes. In an embodiment, the LFA result is ready to read in about 5 minutes to about 30 minutes. In an embodiment, the LFA result is ready to read in about 5 minutes to about 30 minutes. In an embodiment, the LFA result is ready to read in at most about 5, 10, 15, 20, 25 or 30 minutes. [0180] In an embodiment, the LFA device described herein provides high assay specificity resulting in a low rate of false positive results. In an embodiment, high assay specificity is represented by low cross-reactivity with other analytes. Cross-reactivity can result in determination of a positive (early, late, or intermediate) immune status upon analysis of a sample.

[0181] In an embodiment, a low or acceptable level of false positives, resulting from, e.g., cross-reactivity, is represented by an occurrence of cross-reactivity or false positives, at less than 20%. In an embodiment, an acceptable rate or very low rate of cross-reactivity or false positives is represented by an occurrence of cross-reactivity or false positive results, at less than about 20%, preferably less than about 19%, preferably less than about 18%, preferably less than about 17%, preferably less than about 16%, preferably less than about 15%, preferably less than about 14%, preferably less than about 13%, preferably less than about 12%, preferably less than about 11%, preferably less than about 10%, preferably less than about 9%, preferably less than about 8%, preferably less than about 7%, preferably less than about 6.5%, preferably less than about 6%, preferably less than about 5%, preferably less than about 4%, preferably less than about 3%, preferably less than about 2%, preferably less than about 1% or more preferably 0%.

[0182] In an embodiment, the specificity of the LFA is represented by a high correlation of the specificity test results with the results for the same samples obtained in a second assay, e.g., ELISA. In an embodiment, the correlation of the test results with the results for the same samples obtained in a second assay is 90% or greater. In an embodiment, the correlation of the test results with the results for the same samples obtained in a second assay is at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98%, preferably at least about 99%, or more preferably 100%. In an embodiment, the correlation of the test results with the results for the same samples obtained in a second assay is about 80% to 100%. In an embodiment, the correlation of the test results with the results for the same samples obtained in a second assay is at least about 80%. In an embodiment, the correlation of the test results with the results for the same samples obtained in a second assay is preferably about 80% to about 85%, preferably about 80% to about 90%, preferably about 80% to about 92%, preferably about 80% to about 93%, preferably about 80% to about 94%, preferably about 80% to about 95%, preferably about 80% to about 96%, preferably about 80% to about 97%, preferably about 80% to about 98%, preferably about 80% to about 99%, preferably about 80% to about 100%, preferably about 85% to about 90%, preferably about 85% to about 92%, preferably about 85% to about 93%, preferably about 85% to about 94%, preferably about 85% to about 95%, preferably about 85% to about 96%, preferably about 85% to about 97%, preferably about 85% to about 98%, preferably about 85% to about 99%, preferably about 85% to about 100%, preferably about 90% to about 92%, preferably about 90% to about 93%, preferably about 90% to about 94%, preferably about 90% to about 95%, preferably about 90% to about 96%, preferably about 90% to about 97%, preferably about 90% to about 98%, preferably about 90% to about 99%, preferably about 90% to about 100%, preferably about 92% to about 93%, preferably about 92% to about 94%, preferably about 92% to about 95%, preferably about 92% to about 96%, preferably about 92% to about 97%, preferably about 92% to about 98%, preferably about 92% to about 99%, preferably about 92% to about 100%, preferably about 93% to about 94%, preferably about 93% to about 95%, preferably about 93% to about 96%, preferably about 93% to about 97%, preferably about 93% to about 98%, preferably about 93% to about 99%, preferably about 93% to about 100%, preferably about 94% to about 95%, preferably about 94% to about 96%, preferably about 94% to about 97%, preferably about 94% to about 98%, preferably about 94% to about 99%, preferably about 94% to about 100%, preferably about 95% to about 96%, preferably about 95% to about 97%, preferably about 95% to about 98%, preferably about 95% to about 99%, preferably about 95% to about 100%, preferably about 96% to about 97%, preferably about 96% to about 98%, preferably about 96% to about 99%, preferably about 96% to about 100%, preferably about 97% to about 98%, preferably about 97% to about 99%, preferably about 97% to about 100%, preferably about 98% to about 99%, preferably about 98% to about 100%, or more preferably about 99% to about 100%. [0183] In an embodiment, the LFA device described herein provides high assay sensitivity. In embodiments, assay sensitivity is represented by a low or acceptable rate of false negative results. In an embodiment, a low or acceptable rate of false negative results is represented by the occurrence of false negative results at less than 20%, preferably less than 19%, preferably less than 18%, preferably less than 17%, preferably less than 16%, preferably less than 15%, preferably less than 14%, preferably less than 13%, preferably less than 12%, preferably less than 11%, preferably less than 10%, preferably less than 9%, preferably less than 8%, preferably less than 7%, preferably less than 6%, preferably less than 5%, preferably less than 4%, preferably less than 3%, preferably less than 2%, preferably less than 1%, preferably 20% or less, preferably 19% or less, preferably 18% or less, preferably 17% or less, preferably 16% or less, preferably 15% or less, preferably 14% or less, preferably 13% or less, preferably 12% or less, preferably 11% or less, preferably 10% or less, preferably 9% or less, preferably 8% or less, preferably 7% or less, preferably 6% or less, preferably 5% or less, preferably 4% or less, preferably 3% or less, preferably 2%, or less, preferably 1% or less, or more preferably 0%. [0184] In an embodiment, a low or acceptable level of false negative results, is represented by an absence of false negative results of about 80% or greater, preferably about 81% or greater, preferably about 82% or greater, preferably about 83% or greater, preferably about 84% or greater, preferably about 85% or greater, preferably about 86% or greater, preferably about 87% or greater, preferably about 88% or greater, preferably about 89% or greater, preferably about 90% or greater, preferably about 91% or greater, preferably about 92% or greater, preferably about 93% or greater, preferably about 94% or greater, preferably about 95% or greater, preferably about 96% or greater, preferably about 97% or greater, preferably about 98% or greater, preferably about 99%, or more preferably greater 100%.

[0185] In an embodiment, the occurrence of false negative results when using the LFA is 10% or less, preferably 9% or less, preferably 8% or less, preferably 7% or less, preferably 6% or less, preferably 5% or less, preferably 4% or less, preferably 3% or less, preferably 2% or less, preferably 1% or less, preferably less than 10%, preferably less than 9%, preferably less than 8%, preferably less than 7%, preferably less than 6%, preferably less than 5%, preferably less than 4%, preferably less than 3%, preferably less than 2%, preferably less than 1%, preferably 10% or less, preferably 9% or less, preferably 8% or less, preferably 7% or less, preferably 6% or less, preferably 5% or less, preferably 4% or less, preferably 3% or less, preferably 2% or less, preferably 1% or less, or more preferably 0%. [0186] In an embodiment, the sensitivity of the LFA device is represented by a high correlation of the sensitivity test results with the results for the same samples obtained in a second assay, e.g., by ELISA. In an embodiment, the correlation of the test results with the results for the same samples obtained in a second assay is 90% or greater. In an embodiment, the correlation of the test results with the results for the same samples obtained in a second assay is at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98%, preferably at least about 99%, or more preferably 100%. In an embodiment, the correlation of the test results with the results for the same samples obtained in a second assay is about 80% to 100%. In an embodiment, the correlation of the test results with the results for the same samples obtained in a second assay is at least about 80%. In embodiments, the correlation of the test results with the results for the same samples obtained in a second assay is about 80% to about 85%, preferably about 80% to about 90%, preferably about 80% to about 92%, preferably about 80% to about 93%, preferably about 80% to about 94%, preferably about 80% to about 95%, preferably about 80% to about 96%, preferably about 80% to about 97%, preferably about 80% to about 98%, preferably about 80% to about 99%, preferably about 80% to about 100%, preferably about 85% to about 90%, preferably about 85% to about 92%, preferably about 85% to about 93%, preferably about 85% to about 94%, preferably about 85% to about 95%, preferably about 85% to about 96%, preferably about 85% to about 97%, preferably about 85% to about 98%, preferably about 85% to about 99%, preferably about 85% to about 100%, preferably about 90% to about 92%, preferably about 90% to about 93%, preferably about 90% to about 94%, preferably about 90% to about 95%, preferably about 90% to about 96%, preferably about 90% to about 97%, preferably about 90% to about 98%, preferably about 90% to about 99%, preferably about 90% to about 100%, preferably about 92% to about 93%, preferably about 92% to about 94%, preferably about 92% to about 95%, preferably about 92% to about 96%, preferably about 92% to about 97%, preferably about 92% to about 98%, preferably about 92% to about 99%, preferably about 92% to about 100%, preferably about 93% to about 94%, preferably about 93% to about 95%, preferably about 93% to about 96%, preferably about 93% to about 97%, preferably about 93% to about 98%, preferably about 93% to about 99%, preferably about 93% to about 100%, preferably about 94% to about 95%, preferably about 94% to about 96%, preferably about 94% to about 97%, preferably about 94% to about 98%, preferably about 94% to about 99%, preferably about 94% to about 100%, preferably about 95% to about 96%, preferably about 95% to about 97%, preferably about 95% to about 98%, preferably about 95% to about 99%, preferably about 95% to about 100%, preferably about 96% to about 97%, preferably about 96% to about 98%, preferably about 96% to about 99%, preferably about 96% to about 100%, preferably about 97% to about 98%, preferably about 97% to about 99%, preferably about 97% to about 100%, preferably about 98% to about 99%, preferably about 98% to about 100%, or more preferably about 99% to about 100%.

Kits

[0187] The present disclosure also extends to kits comprising the LFA device described herein, optionally in a single housing, such as in a cassette holding the LFA device. In an embodiment, the kit comprises an amount of a buffer (e.g., PBS) sufficient to enable proper flow of the sample analyte(s) and tracer antigen to the test and control regions. For example, where about 40 uL of buffer is sufficient to properly operate each strip, the kit may include at least about 50 uL to about 100 uL of buffer per LFA. Additional kit components may include an instrument for sample collection (e.g., a sharp instrument for drawing blood, or a swab for collecting saliva, urine, semen, or vaginal fluid) and an instrument for applying the sample to the sample application region (e.g., a dropper).

[0188] The assay kit may further comprise instructions for use, which may suitably include a description of test pattern interpretation, and recommendations for subject action based on the result obtained. In another embodiment, the instructions for use include a cautionary warning based on the result interpretation.

[0189] The above references in all sections of this application are herein incorporated by references in their entirety for all purposes.

[0190] All of the features disclosed in this specification (including the references incorporated by reference, including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

[0191] Although specific examples are illustrated and described herein, it will be appreciated by those of ordinary skill in the art that any arrangement calculated to achieve the same purpose could be substituted for the specific examples shown. This application is intended to cover adaptations or variations of the present subject matter. Therefore, it is intended that the invention be defined by the attached claims and their legal equivalents, as well as the following illustrative aspects. The above described aspects and embodiments of the invention are merely descriptive of its principles and are not to be considered limiting. Further modifications of the invention herein disclosed will occur to those skilled in the respective arts and all such modifications are deemed to be within the scope of the invention. Further adaptations of the methods and systems described herein may be accomplished by appropriate modifications by one of ordinary skill in the art without departing from the scope of this disclosure. Several of such potential modifications have been mentioned, and others will be apparent to those skilled in the art. For instance, the examples, embodiments, geometries, materials, dimensions, ratios, steps, and the like discussed above are illustrative and are not required. [0192] These and other features and advantages of various exemplary embodiments of the devices and methods according to this invention are described in, or are apparent from, the following detailed description of various exemplary embodiments of the methods according to this invention.

EXAMPLES

Materials and Methods

1. Specimen Collection and Preparation

[0193] All human specimen materials were considered infectious and hazardous and handled using standard biosafety procedures.

[0194] Plasma: Blood specimen was collected in a lavender or blue top Vacutainer® collection tube containing EDTA or citrate (respectively), using venepuncture. Plasma was separated by centrifugation. Plasma was then carefully withdrawn and decanted into a new pre-labelled tube.

[0195] Serum: Blood specimen was collected in a red top Vacutainer® collection tube without the presence of coagulants. Blood is allowed to clot, and the serum separated using centrifugation. The serum is then carefully withdrawn and decanted into a new pre-labelled tube.

[0196] Specimens were tested as soon as possible after collection. If this was not possible, specimens were stored at 2-8°C for up to 3 days, or frozen at -20°C for longer term storage. For frozen samples, more than 4 freeze-thaw cycles was avoided. Prior to testing, frozen specimens were brought to room temperature slowly and gently mixed. Samples containing visible particulate matter was clarified by centrifugation before testing. Samples demonstrating gross lipaemia, gross haemolysis or turbidity were discarded to avoid interference on result interpretation.

[0197] Whole blood: Drops of blood can also be obtained be venipuncture, or fmgerstick blood. Haemolysed blood is not appropriate for testing. Whole blood specimens are stored at 2-8°C if not tested immediately. 2. Reagents

[0198] Unless stated otherwise, the RBD used in these studies was an N- and C-terminal truncated variant of SEQ ID NO:2, as described elsewhere herein. SARS-CoV-2 RBD-avi (a recombinant RBD that further incorporates a 15 amino acid ‘avi- tag’(GLNDIFEAQKIEWHE)) was produced in-house in mammalian Expi-293F cells (Gibco) by adapting the protocol described in Fairhead and Howarth (2015, Methods Mol. Biol. 1266: 171-184). The SARS-CoV-2 RBD-avi protein was subsequently purified by Ni- NTA purification. SARS-CoV-2 RBD-avi protein was then biotinylated using 5 pg recombinant biotin ligase from E. Coll (BirA protein/lmg of SARS-CoV-2 RBD-avi in Biomix (0.05M bicine buffer, pH 8.3, lOmM ATP, lOmM MgOAc, 50pM d-biotin). Biotinylated SARS-CoV-2 RBD-biotin was purified by running over HiLoad Superdex 75 16/600 (GE Healthcare). Biotinylation was found to be 95% efficient by Streptavidin gelshift.

[0199] The extracellular domains of human ACE2-fiised to the human IgGl-Fc region (Bruce Wines/Mark Hogarth) was produced in Expi-293F cells as above, and purified using Protein A bead slurry (WEHI mAb facility in Bundoora, Australia) and subsequently HiLoad Superdex 200 16/600 (GE Healthcare).

[0200] Anti-RBD monoclonal antibodies were kindly provided by Dr Adam Wheatley (University of Melbourne, Australia). The non-neutralizing anti-RBD monoclonal antibody Antibody 1 is described in Meulen et al. (2006, Pios Medicine, 3(7): e237). The nonneutralizing anti-RBD monoclonal antibody Antibody 2 was isolated from an acute COVID- 19 subject. Briefly, plasmablasts (CD 19+ CD38++ CD27++) were sorted from the patient and the B cell receptor was sequenced using standard multiplex PCR approaches. The nucleic acid sequences encoding the heavy and light chains were synthesised and cloned into human IgGl expression vectors and expressed in Expi293 cells. Antibody 2 was found to bind the RBD of SARS-CoV-2, but did not neutralise SARS-CoV-2 in vitro using the Reed- Muench method, as previously described in Subbarao et al. (2004; J Virol 78:3572-3577). Neutralizing anti-RBD monoclonal antibodies mAb #37 and mAb #42 were recovered from a cohort of convalescent COVID- 19 positive patients. Briefly, patients were pre-screened for serological anti-SARS-CoV-2 neutralising activity, and six patients were selected to sort SARS-CoV-2 spike protein-specific B cells using fluorescent antigen probes. B cell receptor sequences were recovered using standard multiplex PCR, cloned into human IgGl expression vectors and expressed using Expi293 or ExpiCHO expression systems. mAb #37 and #42 were identified as (i) capable of binding to RBD and (ii) potently neutralising using the Reed-Muench method, as previously described in Subbarao et al. (2004; J Virol 78:3572-3577). Anti-biotin-gold (40 nm) was obtained from BBISolutions, UK.

3. Test Principle

[0201] In this embodiment, a lateral flow chromatographic immunoassay was developed that can detect antibodies against the SARS-CoV-2 virus that interfere with binding of the Spike protein (RBD region; SEQ ID NO:2) to the viral receptor, ACE2 protein. The test cassette consists of: 1) a coloured conjugate pad containing SARS-CoV-2 recombinant antigen (Spike RBD proteins; SEQ ID NO:2) conjugated with colloidal gold (SARS-CoV-2 conjugates); 2) a nitrocellulose membrane strip containing an ACE2 line (Test 1 Line) coated with recombinant ACE2, a total antibody line (Test 2 Line) coated with recombinant spike protein RBD, and the control line (C Line) coated with anti-chicken IgY antibody.

[0202] When a correct volume of test specimen (for example, 20 pl) is dispensed into the sample well of the test cassette, the specimen mixes with the SARS-CoV-2 conjugate and migrates by capillary action along the cassette. The anti-SARS-CoV-2 virus antibodies, if present in the specimen, will bind to the SARS-CoV-2 conjugates. If no antibody is present, or if the antibodies bind to RBD protein but without capacity to interfere with ACE2 binding (i.e., lack of neutralization), then the full amount of the SARS-CoV-2 conjugate will bind to the ACE2 line (test line 1). If neutralizing antibody is present in the specimen, then less of the immunocomplex will be captured by binding to the ACE2 protein. In either case, additional SARS-CoV-2 conjugate will flow to test 2 line, where binding of antibodyantigen complexes to the same or related antigen on the test strip will indicate the presence of antibodies to SARS-CoV-2 that are not neutralizing (which is expected even in a subject where there is an abundance of neutralizing antibody), forming a coloured signal, indicating a SARS-CoV-2 total antibody positive test result. Alternatively, test 2 line may be an anti- IgG test line.

[0203] The anti-SARS-CoV-2 RBD antibody, if present in the specimen, will bind to the SARS-CoV-2 conjugates. If the antibodies are able to interfere with RBD-ACE2 binding, then the relevant portion of the immunocomplex will not bind to ACE2 (test 1 line), but will still bind to RBD (test 2 line) via an antigen-antibody-antigen complex (double antigen sandwich). If the antibodies in the immunocomplex are not neutralizing (that is, do not interfere with ACE2 binding) then the SARS-CoV2 conjugate (with or without bound antibodies) will be captured by the ACE2 line, forming a coloured test 1 Line. If this line in not reduced in intensity relative to a pre-determined control (such as the intensity of the control line, Chicken IgY), then it indicates that the patient does not have antibodies that can inhibit RBD-ACE2 binding, indicating lack of potential immunity. If this line is reduced in intensity relative to a pre-determined control (such as the intensity of the control line, Chicken IgY, or a test run in parallel that is not exposed to the test biological sample), then it indicates that the patient DOES have antibodies that can inhibit RBD-ACE2 binding, indicating the presence of potential immunity.

[0204] The test contains an internal control (C Line) which should exhibit a colored band of anti-chicken IgY/chicken IgY- gold conjugate immunocomplex regardless of the color development on any of the test bands (test line 1 and test line 2). If no control band is observed, the test result is invalid and the specimen must be retested.

[0205] The reference values for this lateral flow immunoassay can be easily in line with new knowledge to best reflect the presence of potential immunity to SARS-CoV-2 infection.

4. Test Procedure

[0206] When samples are ready for testing, the test device is removed from packaging and placed on a flat surface, and labelled with specimen ID number.

[0207] A small volume (in this case, 20pL) of serum, plasma or whole blood sample is collected using a transfer pipette and dispensed into the centre of the sample well, ensuring there are no air bubbles. 2 drops of sample diluent buffer are added immediately to the well. [0208] Test readings can be taken in 15 to 20 minutes, either visually by reference between test line 1 and control line, and/or by reading of the test result in a point-of-care test reader such as the AXXIN AX-2XS device.

[0209] This test contains a built-in control feature, the C Line. The C Line develops after addition of the specimen and sample diluent {Valid assay). If the C Line does not develop, the test is invalid {Invalid assay), and the test will need to be repeated.

[0210] The positive and negative controls can be spun down before use. In addition to the presence of C Line, a line at test line 1 should be visible for the negative control and both the test line 1 and test line 2 are visible for the positive controls. The positive control may contain total antibody to SARS-CoV-2 of any isotype but with capacity to inhibit binding of RBD protein to ACE2. Additional controls may be qualified and tested by the user.

5. Interpretation of test result

(a) Valid assay

[0211] In addition to the presence of the C Line, if only the Test Line 1 is developed, the test result indicates the absence any patient antibody to RBD of SARS-CoV-2 virus. The result is non-reactive, consistent with no immunity to the virus and potentially lack of any prior or current infection with the virus.

[0212] In addition to the presence of the C Line, if test line 1 develops with lower intensity, where no antibody is present, as determined by comparison to the C line or using an instrument such as the Axxin AX-2XS instrument, and test line 2 develops with any level of intensity, then the test indicates the presence of anti-SARS-CoV-2 virus antibody that can inhibit RBD-ACE2 interaction and is therefore consistent with the presence of neutralizing antibody. The result is neutralizing antibody positive or reactive, consistent with some level of potential immunity to SARS- CoV-2 virus infection.

[0213] In addition to the presence of the C Line, if test line 1 develops with the same intensity as in the case where no antibody is present, as determined by comparison to the C line or using an instrument such as the Axxin AX-2XS instrument, and test line 2 develops with any level of intensity, then the test indicates the presence of anti-SARS-CoV-2 virus antibody but the absence of antibody that can inhibit RBD-ACE2 interaction consistent with neutralizing antibody. The result is neutralizing antibody negative or non-reactive, consistent with minimal or no potential immunity to SARS- CoV-2 virus infection.

(b) Invalid assay

[0214] If the C Line does not develop, the assay is invalid regardless of color development of the test line 1 and test line 2 as indicated below. In such cases, the assay should be repeated with a new device.

6. Proof of Principle assay

[0215] The capture antigen or protein (recombinant human ACE2 receptor attached to the Ec portion of immunoglobulin; referred to as Fc-ACE2) is immobilised on to nitrocellulose strips in uniform bands of 2mg/mL and 0.8mg/mL. Strips contain a sample pad at the bottom to aid sample loading and an absorption pad at the top to wick the analyte up by capillary action.

[0216] The receptor binding domain of SARS-CoV-2 spike protein (SEQ ID NO:2) tagged with biotin (known as RBD-biotin) is labelled with anti -biotin antibodies conjugated with gold particles (which give a red colour) by incubating together for a period of time, e.g. 5 minutes.

[0217] Sample serum to be interrogated is mixed with the gold-labelled RBD for a period of time, e.g. 10 minutes, to allow (if present) neutralising anti -RBD antibodies in the serum to bind to the SARS-CoV-2 RBD.

[0218] The sample pad of a strip of immobilised Fc-ACE2 is then placed in the gold-labelled RBD/serum mixture for 10 min, drawing the solution along the strip. Once the sample has been loaded on to the strip, the strip is then placed in a wash buffer (containing PBS/0.5% Tween20) for a further 10 min to remove excess gold-labelled RBD. Strips are then visually assessed or analysed on the Axxin machine for a diminution of the red test line indicating the interaction of Fc-ACE2 and gold-labelled RBD has been inhibited by the presence of neutralising anti-RBD antibodies in the sample.

7. Production of Lateral flow device

[0219] The device cassette consists of a plastic housing (Nanjing BioPoint Diagnostics, PR China) with loading wells and a window to read results. Within the cassette is a nitrocellulose membrane strip with, at one end, (A) a sample loading pad, (B) a conjugate pad containing: 1) biotinylated RBD protein complexed with anti -biotin- colloidal gold (BBISolutions, UK), (SARS-CoV-2 conjugate); 2) colloidal gold-conjugated chicken IgY as a control and reference standard and (C) a mixing pad. The mixing pads are arranged sequentially so that sample flows from the sample pad, to the conjugate pad, to the mixing pad, then to the nitrocellulose membrane.. The nitrocellulose membrane strip has three lines or stripes of test reagents, the first of which is a recombinant chimeric protein consisting of the extracellular domain of human ACE2 fused to human-IgGl-Fc domain line (test line 1), a control/reference line (Ref Line) coated with anti -chicken IgY antibody and a total anti-RBD antibody line (Test Line 2) coated with recombinant RBD protein. [0220] The test specimen ( 15 pl plasma) is dispensed into the sample well of the test cassette, and three drops of running buffer (phosphate buffered saline pH 7.4) are added to well B of the test cassette. For the test modified for whole blood, the test specimen (3 Opl) and running buffer are added to the same well. In both cases, the specimen mixes with the colloidal gold RBD conjugates upon sample addition and during migration by capillary action in the mixing pad, and then migrate along the nitrocellulose membrane. Anti-SARS-CoV-2 RBD antibodies, if present in the specimen, will bind to the RBD-biotin-Au conjugates. If no antibody is present, or if the antibodies bind to RBD protein but without capacity to interfere with ACE2 binding (i.e., lack of neutralization), then the maximum amount of RBD-gold will bind to the ACE2 line (test line 1). If neutralizing antibody is present in the specimen, less of the immunocomplex will bind to the ACE2 protein. In either case, as the sample continues to flow, the chicken IgY-gold will bind to the anti -chicken IgY line (control/reference line), and next, additional SARS-CoV-2 conjugate will flow to test line2, where binding of antibody-antigen complexes to the same or related antigen on the test strip will indicate the presence of antibodies to SARS-CoV-2 that may or may not be neutralizing because ACE2 is not involved in this interaction. This indicates a SARS-CoV-2 RBD antibody positive test result.

[0221] The intensity of the control/reference line can be adjusted by varying the amount of anti-chicken IgY on the nitrocellulose and/or the amount of chicken IgY -gold at the time of manufacture, in order to reflect the best estimates for protective levels of antibody for immunity to SARS-CoV-2 infection, such as 70% inhibition, or 50% inhibition, or 30% inhibition, or other levels as desired. The RBD-biotin can be expressed using any variant sequence of RBD and is then titrated to determine an equivalent level of binding to that seen for the reference RBD-biotin for the inhibition assay.

[0222] To determine minimum of the amount of ACE2-biotin required to detect I pg of immobilised RBD-Fc, Ipg of RBD-Fc immobilised onto the strip was exposed to 30 pL of 5 different concentrations of ACE2-biotin (50, 5, 0.5, 0.05 0.005 pg/mL), and any bound ACE2-biotin is detected using gold-conjugated anti-biotin antibody, as described in the preceding paragraph. 5pg/mL appears to be the limit. (Figure3). This suggests that the optimal concentration of ACE2 -biotin is between 5 and 25pg/ml. 8. Microneutralisation assay

[0223] Microneutralisation assays were performed as previously described (Koutsakos, M. etal., 2021, Cell Rep. Med. 2: 100208; Juno, J. etal., 2020; Nat. Med.; 26, pages 1428- 1434). SARS-CoV-2 isolate CoV/Australia/VICO 1/2020 (Caly, L. et al., 2020, Med. J. Aust., 212 (10): 459-462) was passaged in Vero cells and stored at -80C. Plasma was heat inactivated at 56 °C for 30 min. Plasma was serially diluted 1:20 to 1: 10,240 before the addition of 100 TCID50 of SARS-CoV-2 in MEM/0.5% BSA and incubation at room temperature for 1 h. Residual virus infectivity in the plasma/virus mixtures was assessed in quadruplicate wells of Vero cells incubated in serum-free media containing 1 pg ml^-1 of TPCK trypsin at 37 °C and 5% CO2; viral cytopathic effect was read on day 5. The neutralizing antibody titre was calculated using the Reed-Muench method, as previously described (Subbarao, K. et al., 2004, J. Virol., 78(7):3572-7).

9. Statistics

[0224] Regression lines fitted in GraphPad Prism 9.0 using a semi-log non-linear fit, plotted with 95% CI bands. R² goodness-of-fit displayed on graphs. For comparison of multiple groups, Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparison test.

Example 1 - Detection of immobilised SARS-CoV-2 Receptor binding domain

[0225] For initial titration of how much SARS-CoV-2 Receptor Binding Domain (RBD) is required for visual detection on the strip, equal volumes of three different concentrations of RBD-Fc / RBD-biotin (0.01, 0. 1 and 1 mg/mL) were spotted onto the strip, dried at 37°C for 15 minutes. The RBD was the truncated protein of SEQ ID NO:2. The strips were then incubated in 30pL of their respective gold-conjugated anti-IgG or anti-biotin antibodies for 10 minutes, and then washed with 30pL running buffer (Figure 1).

[0226] To titrate the amount of biotin-labelled Angiotensin Converting Enzyme 2 (ACE2- biotin) required to detect 1 pg of immobilised RBD-Fc, 1 pg of RBD-Fc was immobilised onto the strip and exposed to three different concentrations of ACE2 -biotin (25, 50 and 100 pg/mL) for 10 minutes. A control using just blank running buffer is included (bottom strip). The samples are washed with 30pL running buffer, and then exposed to either 30pL gold- conjugated anti-biotin antibody (for ACE2 spots) or gold-conjugated anti-IgG for another 10 minutes. The strips are washed with running buffer. This indicated that ACE2 -biotin could bind to immobilised RBD giving a similar signal for each of the concentrations tested down to 25pg/ml. (Figure 2).

[0227] To determine minimum of the amount of ACE2-biotin required to detect 1 ig of immobilised RBD-Fc, Ipg of RBD-Fc immobilised onto the strip was exposed to 30 pL of 5 different concentrations of ACE2-biotin (50, 5, 0.5, 0.05 0.005 pg/mL). and any bound ACE2-biotin is detected using gold-conjugated anti-biotin antibody, as described in the preceding paragraph. 5pg/mL appears to be the limit. (Figure3). This suggests that the optimal concentration of ACE2 -biotin is between 5 and 25pg/ml.

Example 2 - Titration of uninfected and COVID-19 human sera samples

[0228] This experiment was undertaken to test if sera from healthy subjects (i.e. with no known prior infection with SARS-CoV-2) could blocked binding of ACE2 to RBD. Using the same experimental set-up described above, including I pg of RBD (SEQ ID NO:2)-Fc immobilised onto the strip, the strips were exposed to ACE2 -biotin that was pre-incubated with 80%, 16%, 3.2%, 0.64%, 0. 128% and 0.0256% dilutions of healthy human serum. The 80% dilution sample consisted of neat human serum sample with gold-conjugated anti-biotin and ACE2. Only at the highest (80%) diluted serum resulted in any reduction of the signal development (Figure 4). This suggested that healthy human serum should be used at concentrations below 80% in order to avoid non-specific inhibition of binding of ACE2- biotin to immobilised RBD-Fc.

[0229] Having established the basic conditions that allowed detection of ACE-2 bound to RBD-Fc, strips were made with a dedicated striping machine. When the strip with Ipg of immobilised RBD-Fc was incubated with 20% dilution of serum samples from a healthy subject (206) or from a COVID-19 patient (302), before being exposed to ACE2-biotin, and detected, as described above, some apparent inhibition of signal development could be observed (Figure 5). This indicated that COVID-19 patient sample 302 could inhibit the binding of ACE2 -biotin with immobilised RBD-Fc, but the reduction in signal was not very strong.

[0230] Because the reduction in signal was not very strong, it was reasoned that there may be too much RBD striped on the strip. Therefore, three different amounts of RBD-Fc (1.0, 0.3 and O. lug/ml) striped onto strips were tested. These strips were tested at three different concentrations of ACE2-biotin (5.0, 0.5, 0.05pg/ml). (Figure 6). These results showed a clear titration of ACE2 -biotin that was detectable down to 0.5pg/ml, for each of the RBD concentrations. However, the signal grew fainter as the RBD concentration decreased. Because we want a signal that is clearly detectable, but not too strong and more difficult to inhibit, these data suggested that 0.3ug/ml RBD striped strips and 0.5pg/ml ACE2 was ideal for further assays.

[0231] In order to increase the amount of antibody in the samples tested, we tested a higher percentage of serum. Using the conditions established in Figure 6, we tested a range of healthy human serum concentrations (0, 10, 20 and 40%) to determine what is the highest amount we can use before we see non-specific inhibition of ACE2-RBD binding. Two approaches were used: 1. Where the human serum was pre-run on the strips, followed by ACE2-biotin mixed with anti-biotin-gold; or 2. Where the human serum was mixed with ACE2-biotin and anti-biotin-gold and they all were run together. These data (Figure 7) showed that up to 40% serum did not interfere with the ACE2-RBD interaction providing the serum was pre-run before the ACE2. If the serum was mixed with ACE2, then only 10% serum could be tolerated.

[0232] Using the new conditions established, we tested the ability of at least 40% serum from a COVID- 19 patient to inhibit RBD-ACE2 binding. Three different formats were tested (Figure 8): 1. 0.3pg/ml RBD-Fc stripe, ran 40% serum, detected w 0.5pg/ml ACE2; 2. O. lpg/ml RBD-Fc stripe, ran 40% serum, detected w 0.5pg/ml ACE2; 3. 0.3pg/ml RBD-Fc stripe, run 10% serum + ACE2 -biotin + anti -biotin-gold. 40% COVID- 19 patient serum seemed to cause a stronger reduction in the signal compared to 10% serum, suggesting that the amount of antibody in serum is limiting.

[0233] Further, COVID- 19 patient sera samples were analysed. Strips with Ipg of immobilised RBD-Fc were incubated with 60% dilution of serum samples from healthy uninfected patients (samples 36-50) or COVID- 19 patients (samples 302-345) before being exposed to ACE2-biotin (Figure 9). The signals were quantified, and some reduction in the signal could be observed with the serum samples from the COVID-19 patients compared to the samples from healthy donors. These samples were read on an Axxin strip reader instrument giving numerical values for stripe intensity. These data are also graphed as % inhibition based on inhibition of the mean signal intensity from the healthy control samples. This result confirms that COVID- 19 patient samples could inhibit the binding of ACE2- biotin to immobilised RBD-Fc, suggesting the presence of neutralising antibodies. [0234] For a comparison, the same patient samples were assessed for the presence of anti- RBD antibodies. This is not an inhibition assay, and cannot discern the presence of neutralising versus non-neutralising antibodies - it can only demonstrate that anti-RBD antibodies are present. Strips with immobilised RBD-Fc were exposed to 10% dilution of healthy uninfected human sample (36-50) or COVID-19 patient (samples 302-345) patient serum. A control strip was exposed to no patient serum. In the absence of serum, we find that the gold-conjugated anti-human IgG binds to the RBD-Fc directly. This does not happen in the presence of serum. Thus, antibodies to RBD of SARS-CoV-2 in COVID-19 patient serum, but not healthy serum samples, could be detected using gold conjugated goat-anti- human IgG. The presence of these anti-RBD antibodies was independently validated. Antibodies to RBD of SARS-CoV-2 in COVID-19 patient serum could be detected using gold conjugated goat-anti-human IgG. Healthy patient samples did not show presence of anti-RBD antibodies.

Example 3 - Detection of immobilised ACE2

[0235] Attempts were also made to immobilise ACE2 to the strip for use as the capture antigen (receptor) at the test region of the LFA device / test strip, noting that in the previous assays above, serum is run over the RBD before ACE2 is added. Any excess anti-RBD antibody would flow past the RBD strip and ACE2 that follows would be competing only with the antibody that had already bound the RBD so that system is may be less sensitive to higher titres of antibody. If ACE2 is immobilised on the strip and the serum is mixed with the RBD directly while they run across ACE2, the anti-RBD antibodies will potentially be in excess in high titre samples, giving a better measure of the amount of neutralising antibody in a sample. Thus, equal volumes (IpL) of three different concentrations of ACE2-biotin (0.028, 0.28 and 2.8 mg/mL) were spotted onto the strip. However, direct detection attempted with gold conjugated anti -biotin antibody did not reliably detect the loaded protein (Figure 11). This result showed that ACE2 -biotin does not immobilise very effectively to the strip and appears to diffuse away.

[0236] The experiment was repeated with Ipg of ACE2 -biotin or just ACE2 protein without a biotin tag spotted onto the strip and exposed to 3 different concentrations (25, 50 or 100 pg/mL) of RBD-Fc. A control experiment was run where a strip with ACE2-biotin was incubated with blank running buffer. The interaction was assayed using gold conjugated anti-IgG for the test strips or gold conjugated anti-biotin for the control strip was used to detect for the binding of RBD-Fc to the strip. No binding was observed, indicating that no RBD-Fc was bound. The control strip had a signal, showing anti-biotin antibody interacting with ACE2 -biotin suggesting that ACE2 -biotin had bound to the strip. This suggested that RBD-Fc could not bind to strip-bound ACE2-biotin, suggesting that strip-bound ACE2- biotin had somehow changed its conformation (Figure 12).

[0237] An attempt to immobilisation ACE2 -biotin to the strip was made using streptavidin to capture ACE2 -biotin on strip, since the previous experiments suggested that it did not retain the ability to bind to RBD when it was directly immobilised to the strip. Equal volumes of streptavidin (SAV) or streptavidin- Phycoerythrin (SAV-PE) of different concentrations were spotted onto the strip, dried at 37°C for 15 minutes, before being incubated with biotin. The streptavidin-biotin complex was then detected using gold-conjugated anti-biotin antibodies. Ipg streptavidin could be detected, indicating that streptavidin could be immobilised to the strip (Figure 13A). Ipg streptavidin spot/strip was then incubated with 30pL ACE2 -biotin (50pg/mL), before being washed with 30pL running buffer. The strips were then exposed to 3 different amounts of RBD (0, 0.5, 5 and 50 pg), and detected using gold-conjugated anti-IgG antibodies. This resulted in successful detection (formation of signal) (Figure 13B), suggesting that ACE2-biotin can be immobilised to the strip using streptavidin. RBD binding could be detected at each concentration but 5ug appeared to be optimal, whereas 0.5 pg was very faint.

Example 4 - Immobilised ACE2-biotin and inhibition assays using COVID-19 patient samples

[0238] Using streptavidin-ACE2 -biotin immobilised spots, two human COVID- 19 sera samples (322-B and 325-B) or two healthy control sera samples (039, 046), each diluted at 60% and run on the strip mixed with 5ug/ml RBD-Fc. A no sera control was also included. While the intensity of the signals were very faint, even in the absence of sera or in presence of sera from healthy controls, inhibition was observed with the COVID- 19 sera samples. (Figure 14A).

[0239] With encouragement from this experiment, we had some strips striped up with streptavidin so we could get the strips read on the Axxin strip reader. The experiment was repeated in a similar manner to A, using streptavidin striped strips, and a panel of 11 healthy human sera samples and a panel of 10 COVID- 19 patient sera. Unfortunately, we found that the signals even from the control samples were very weak and they also diffused/faded very quickly suggesting the ACE2 was not well-bound to the strips. This made it very difficult to judge if we were detecting inhibition of signal in presence of COVID- 19 patient sera.

[0240] To test if immobilised ACE2 -biotin binding of RBD-Fc can be inhibited with soluble ACE2, streptavidin immobilised ACE2-biotin was exposed to RBD-Fc pre-incubated with soluble, un-biotinylated human ACE2 at different molar ratios (note: there are two RBD units / molecules per RBD-Fc fusion protein). Here we reverted back to the spotting technique for binding ACE2-biotin to streptavidin spotted strips. Anti-mouse Ig-Au was used to identify captured RBD. Excess soluble human ACE2 prevented RBD-Fc binding to immobilised ACE2-biotin. Some dosage -dependent signal inhibition could be observed (Figure 15). However, the intensity of spots was low (even in samples without competing ACE2), and contrast had to be adjusted to give best visual representation.

Example 5 - Immobilisation of Fc-ACE2 allows efficient dose-responsive visualization of RBD binding.

[0241] A different method to immobilise ACE2 was utilised with Fc-ACE2 protein. Different amounts (4, 2, 1, 0.5 0.25 and 0 pg) of ACE2 fused to Fc were spotted onto the strip and successfully detected using gold-conjugated anti -human IgG. This unexpected result indicates that Fc-ACE2 adheres to the strip well (Figure 16A).

[0242] 2pg of Fc-ACE2 was spotted on to the strip and dried at 37°C for 15 minutes, and then exposed to 3 different concentrations of biotinylated RBD (RBD-biotin). Detection of bound RBD-biotin was visualised using gold-conjugated anti-biotin antibody. The signal showed a clear dose-response change in intensity that correlated with the titrated amount of RBD-biotin used (Figure 16B).

[0243] The experiment was repeated using spots of either 2pg or 0.26pg of Fc-ACE2, which were then exposed to 2 concentrations of RBD-biotin (2.4pg/mL or 24pg/mL). A clear signal was seen for the 2pg of Fc-ACE2 spot while a very faint signal could be seen for 0.26pg of Fc-ACE2. In each case, they did not show a dose-response to RBD-biotin suggesting that the lower dose of 2.4pg/ml RBD biotin was saturating (Figure 16C). Thus, using less Fc- ACE2 (0.26 pg) on the strip resulted in some loss of RBD-biotin detection (data not shown) and this suggested that the best dose of ACE2 bound to strip would fall between 0.26pg and 2pg.

Example 6 - Immobilised ACE2-Fc and inhibition assays using COVID-19 patient samples

[0244] Using Fc-ACE2 immobilised spots, sera samples from healthy uninfected patient samples and COVID- 19 patient samples were used to test if the Fc-ACE2 configuration could detect inhibition of binding between ACE2 and RBD.

[0245] To determine if high concentrations of healthy sera could non-specifically interfere with the Fc-ACE2-RBD interaction, Fc-ACE2 was immobilised on the strip and then exposed to RBD-biotin that was pre-incubated with healthy human sera of increasing concentrations (0-80% diluted sera, and a negative control of a 80% diluted sample with no RBD). No signal inhibition could be seen, even at 80% serum dilution. The serum sample with no RBD control experiment gave no signal (Figure 17).

[0246] In the next experiment, the 1 pg Fc-ACE2 spot was pre-incubated with RBD-biotin mixed with either 80% dilution of healthy, uninfected human sera or COVID- 19 human patient sera (independently confirmed to have neutralising antibodies). Detection of RBD- biotin bound to immobilised Fc-ACE2 using gold-conjugated anti-biotin antibodies indicated that the COVID-19 patient sera clearly inhibited signal formation, confirming the presence of neutralising antibodies that blocked binding of ACE2 to RBD (Figure 18).

Example 7 - Titration of RBD-biotin

[0247] To ensure that there was no biotin (vitamin B7) in the serum of COVID-19 patients that might interfere with results of the test, the experiment was modified to saturate the RBD- biotin with anti-biotin-Au antibodies. 1.5 pg of RBD-biotin was spotted on to the strip, and allowed to dry at 37°C for 20 minutes. 30pL of gold-conjugated anti-biotin antibody was flowed onto the spot for 10 minutes, then washed with 60 pL of wash buffer for another 10 minutes. Signal intensity was measured (pre-sera). 30pL of healthy uninfected human sera or COVID- 19 patient sera was then flowed onto the spot, and washed with 60 pL of wash buffer. Signal intensity was measured (post-sera). There was no significant decrease in signal intensity (measured by Image J software) pre or post sera, in either healthy uninfected patient and COVID-19 patient samples (Figure 19). [0248] Titration of RBD-biotin amounts to optimise signal to noise ratio, two lines of Fc- ACE2 was immobilised on two separate lines (test regions) on the strip, at different concentrations (2mg/mL, and 0.8mg/mL). 30uL of 4 different concentrations of RBD-biotin (50, 16.7, 5.5, 1.8 and 0 pg/mL) were flowed across the Fc-ACE2 lines, washed with 30pL of PBS/0.5% Tween wash buffer for 10 minutes, before incubation with 30uL anti-biotin- Au for lOmin, and a final wash with 30uL PBS/0.5% Tween wash buffer (Figure 20A). Quantification of the signals can be seen in graph depicted in Figure 20B.

Example 8 - Proof-of-concept principle using immobilised Fc-ACE2 and RBD-biotin to discern between sera with and without neutralising antibodies.

[0249] RBD-biotin (5 pg/mL) and anti-biotin-gold were pre-mixed and incubated for 5 minutes. To lOpL of these samples, 17pL of buffer and 3pL of sera samples were added and incubated for 10 minutes. There were 5 sera samples: 1) Healthy uninfected human serum sample, 2) COVID- 19 patient serum sample, 3) healthy unimmunised mouse serum sample (healthy mouse (1.5)), 4) mouse serum sample from SARS-CoV-2-RBD immunised mouse (mouse (4.2)) and 5) pre-bleed sample of mouse 4.2 (that is sample taken before immunisation with RBD). Mouse 4.2 was independently shown to have developed high titres of RBD reactive antibodies following immunisation with RBD. These samples were flowed onto strips with 2 test regions of immobilised Fc-ACE2 (at 2mg/mL and 0.8mg/mL concentrations) for 10 minutes, and then washed with 60 pL of PBS/0.5% Tween wash buffer for lOmin. Signal development was then quantified. The 0.8mg/mL ACE2 band gave only a faint signal. However, the 2mg/mL ACE2 test region showed visible signal reduction with the COVID- 19 patient sample, and similar reduction was seen with the RBD immunised mouse serum sample (Figure 21).

[0250] The RBD immunised mouse sample (mouse 4.2) and a healthy unimmunised mouse sample (mouse 1.5) were used to titrate the amount of RBD-biotin necessary for this assay. Approx. 4 pL of 2, 1, 0.5 pg/mL RBD was immobilised onto the strip, pre-incubated with gold-conjugated anti-biotin antibodies, as described above, before incubation with the different serum samples. The opportunity was also taken to compare different dilutions of anti-RBD-antibody containing sera. Only the RBD-containing serum samples gave visible reduction in signal, and this was true of both 20% and 5% dilutions of the serum sample (Figure 22). Example 9 - Application on a panel of human serum samples

[0251] A panel of human plasma and serum samples were analysed using the test assay and protocol described above (this time with a 50% serum dilution due to limited volume of some samples). The (previously determined) COVID-19 status of these samples were not disclosed at the time of the assay and analysis in order to run a blind experiment. Test regions on the strip corresponding to 2 mg/ml Fc-ACE2 (high) and 0.8 mg/ml Fc-ACE2 (low) were used in the assay and the signal quantified using a point-of-care test reader such as the AXXIN AX-2XS device.

[0252] The results are depicted in Figure 23. After the experiment was performed the samples were unblinded and we found that there were only two samples that lacked RBD- specific antibody (independently determined) boxed in red (CVD 07 IB and CVD 307). The quantification data is plotted in Figure 23B displaying peak signal intensity for the 2mg/ml Fc-ACE2 stripe in order from strongest (least inhibited) to weakest (most inhibited). Similar results were determined for the 0.8mg/ml Fc-ACE2 lines. The two samples that lacked RBD antibodies are encircled and their anti-RBD antibody titres shown in red (independently determined). Anti-RBD antibody titres (independently determined) for the most inhibited samples (on the right side of the graph) are also shown in red text in Figure 23B.

[0253] The two independently confirmed COVID- 19-negative samples, CVD 071B (marked as 152 in Figure 23B) and CVD 307 (marked as 123 in Figure 23B) showed two of the strongest intensity signals. Samples known to have higher levels of anti-RBD antibodies showed low levels of signal intensity: CVD342B (anti-RBD titre = 142811 marked in Figure 23B) CVD110B (anti-RBD titre = 119865 marked in Figure 23B), CVD333B (anti-RBD titre = 30512 marked in Figure 23B), CVD318B (anti-RBD titre = 96734 marked in Figure 23B), CVD308B (anti-RBD titre = 13087 marked in Figure 23B).

[0254] After learning that there were only two negative samples in this experiment, we performed an additional experiment (Figure 23C) with additional samples including 10 healthy control serum samples, 8 additional COVID- 19+ samples, plus 4 samples from the experiment shown in Figure 23A in order to normalise between the two experiments. The collective data derived from the experiment in Fig 23 A and Fig 23 C were normalised and used to generate a graph showing the % of RBD-ACE2-binding inhibition in COVID-19- positive and -negative samples where 0% is determined by the mean signal intensity of the healthy control samples, as shown in Fig 23D.

Example 10 - Lateral Flow Assay for sensitive and specific detection of anti-SARS- CoV-2 neutralising antibodies

[0255] The assay was repeated using four anti-RBD monoclonal antibodies, only two of which are known to be neutralising antibodies (Antibodies A and B), while the other two are non-neutralising antibodies (independently determined) at different serial 3-fold dilutions (10: 1, 3.33: 1, 1.11: 1, 0.37: 1, 0.12: 1, and 0.04: 1 molar ratio of antibody :RBD) (Figure 24A). RBD-biotin was pre-incubated with gold-conjugated anti-biotin antibodies, before the addition of the antibodies at the desired concentrations. These samples were incubated for 10 minutes before being flowed over the Fc-ACE2 bound strips, and washed in 60pL wash buffer. The non-neutralising anti-RBD antibodies (Antibody 1 and 2) did not prevent signal formation in a dose dependent manner. Incubation with increasing amounts of neutralising antibodies A and B (mAb #37 and #42) resulted in inhibition of signal formation in a dose dependent manner, indicating their ability to block RBD-ACE2 interaction (Figure 24A). The signal intensity was quantified in the graph in Figure 24B. These data were also quantified using an Axxin reader, shown in Figure 24C.

[0256] After the initial titration experiments ACE2 was applied at 2mg/ml (0.5 pg of ACE2- Fc per 5 mm wide test strip) and this detected a clear visible signal from binding of RBD- Au at 1 pg/ml (16.66 pl of OD3.0 anti-biotin gold mixed with 0.016 pg of RBD-biotin per test strip) (see Figure 25B). Titration of the RBD-biotin before mixing with this fixed amount of anti -biotin gold, yielded a clear visual signal over a range of concentrations from 0.06- 2ug/ml (Figure 25C), with evidence of a “prozone” effect at the highest RBD-biotin concentrations and a linear titration at lower concentrations, allowing us to identify the appropriate level of RBD-biotin that would be readily titratable upon inhibition by neutralising antibodies. It was established an optimal concentration of 2pg/ml ACE2-Fc to coat the assay strip and a maximum of 1 pg/ml RBD-biotin mixed with the anti-biotin gold. This provided a clear dose-response curve showing near complete inhibition of the visual ACE2-RBD-Au signal with the neutralising anti-SARS-CoV-2 monoclonal antibodies mAb #37 and #42, while the non-neutralising mAb had no clear impact on the strength of the signal as shown in Figure 24C. Example 11 - Testing COVID-19 patient samples

[0257] Having established that this assay can detect the presence of neutralising antibodies, 79 COVID-19 patient plasma samples and 47 COVID- 19 negative control samples as well as samples from patients with other virus infections were tested (Figure 26). As expected, the pre-COVID-19 healthy control samples showed minimal fluctuation in the intensity of the RBD-ACE2 signal, within a range of ±20% around the mean. In contrast, COVID-19 patient samples showed a broad range of inhibition in the intensity of the RBD-ACE2 test line, with some samples almost completely inhibiting the signal (Figure 26A). These samples were measured using the Axxin strip reader and the % inhibition within the COVID-19± samples relative to the COVID- 19- samples was calculated as: % inhibition = (1 - sample intensity/median intensity of COVID-19-ve samples) xlOO. Samples taken from patients with other virus infections including RSV, influenza and picomaviruses all showed no inhibition greater than 20% compared to control COVID-19 -ve samples (Figure 26B). These inhibition values were plotted against the neutralising titres from the same samples determined using a SARS-CoV-2 microneutralisation assay, revealing a strong correlation in neutralisation (R²=0.72) (Figure 26C).

Example 12 - Development and testing of a prototype assay for point-of-care use.

[0258] To facilitate intended use of the test in a point-of-care setting, the assay strip configuration was modified to allow the use of a self-contained cartridge (Figure 27). In this LFA cartridge design, a visual reference/control line is incorporated to allow direct estimation of % inhibition within the individual test strip/cartridge, without the need for performing additional controls in separate tests. The intensity of this control/reference line can be adjusted during manufacture by altering the concentration of the respective reference line (in this case, anti -chicken IgY) and visual detector (in this case, chicken IgY -colloidal gold), giving a consistent signal independent of the sample added to the test. As such, the line intensity can be chosen to represent any desired level of inhibition compared to what is observed using representative negative control samples that have no inhibition. In the example referred to in Figure 27, the reference line is set at 50% of the average intensity of representative healthy pre-COVID-19 samples, so that a patient sample giving the same test line intensity as the reference therefore has 50% inhibition; a test line stronger than the reference has <50% inhibition and a test line twice as strong as the reference line has 0% inhibition; a test line weaker than the reference line has >50% inhibition and the absence of a test line represents 100% inhibition.

[0259] In this configuration, a small volume of plasma is added to well A, where the sample rehydrates the RBD-Au complex and begins to migrate into the mixing pad. Three drops of running buffer (buffered saline) are then added to well B, and the buffer chases the plasma and RBD-Au complex mixture along the length of the test strip within the closed cassette. The sample pad also contains a defined amount of control/reference material, gold conjugated chicken IgY, which serves both as an assay running control and as a reference line. The sample then reaches the immobilised ACE2-Fc line, where the amount of RBD- gold complex binding will be inversely related to the amount of neutralising antibody in the sample. Two additional lines have been incorporated on these strips. The reference line is a stripe of anti-chicken IgY that binds to the chicken IgY-gold, which serves as a control to show that the test has run successfully and also serves to provide a reference line level of predetermined intensity for comparison to the ACE2-RBD-Fc stripe where a predetermined amount of inhibition (e.g., 50%) is desired. The intensity of this line can be adjusted at manufacture by varying the amount of anti-chicken IgY and/or chicken IgY-gold that is added, allowing a direct visual comparison between the test and reference lines to assess whether a sample has sufficient levels of neutralising antibody. The third line is striped with RBD protein, which forms a double-antigen sandwich with RBD-gold anti-RBD antibody derived from the sample being tested. This line therefore indicates the presence of total anti- RBD antibody, regardless of whether or not it is neutralising and capable of blocking RBD- ACE2 interaction.

[0260] In this further prototype assay, the quantitative level of % inhibition is calculated by reference to the intensity of the control / reference line. For the examples shown, the reference line was set at a level equivalent to 50% of the mean reactivity for pre-COVID controls, and the % inhibition is calculated according to the formula % inhibition = (I - (test line intensity/(2x reference line intensity)) xlOO. It will be appreciated that the calculation (2x reference line intensity) in this formula can be adjusted according to different reference lines, for example a reference line representing 75% inhibition (i.e. 25% of signal without inhibition) would give the formula % inhibition = (1 - (test line intensity/(4x reference line intensity)) xlOO. [0261] The included reference line also allows the visual, semi-quantitative determination of % inhibition relative to the threshold level of the reference line, without the need for an instrument reader or other equipment. In these examples where the reference line represents 50% inhibition, a test line equal to the reference line would represent a sample with 50% inhibition; a test line of lower intensity than the reference line would represent a sample with >50% inhibition; a sample with higher intensity than the reference line would represent a sample with <50% inhibition. A similar method of semi-quantitative threshold determination is used in the Visitect® CD4 and Visitect® CD4 Advanced Disease T-cell tests (Omega Diagnostics, UK) developed by some of the inventors, where the reference line represents a level of either 350 or 200 CD4 T-cells per microlitre (respectively), and a test line stronger than the reference line indicates T-cell sufficiency (>350 or >200 CD4 T-cells/pl), while a test line weaker than the reference line indicates T-cell insufficiency (<350 or <300 CD4 T- cells/pl).

[0262] This prototype assay was used to examine 72 samples from healthy pre-COVID controls and 200 samples from CO VID- 19 patients, examples of which are shown in (Figure 27C). These data showed a high degree of consistency across the 50 control samples and a wide range of inhibition from 0-95% in the patient samples (Figure 27D). When these data were plotted against neutralising titre data from the same samples determined by microneutralisation assay, a clear correlation was observed (R² = 0.69) (Figure 27E).

[0263] It has been suggested that neutralising antibody levels decrease quite rapidly, within weeks to months of infection, which is an important reason why tests that can measure neutralising antibody may be necessary to confirm adequate levels of neutralising antibody at regular intervals after infection or vaccination. We examined a series of longitudinal samples from a single subject (Cl) beginning 3 weeks after confirmed PCR diagnosis of infection with SARS-COV-2 (Figure 27F). Data from another subject (C2.1) is also included. These data show that over the period of 3-8 weeks, the degree of ACE2-RBD inhibition shown in the test line declined from ~70% to 18.1%, in parallel with declining levels of total anti-RBD Ig. In contrast, a single sample from patient C2. 1 shows high levels of total anti-RBD Ig but negligible (3%) ACE2-RBD inhibition (Figure 27F), highlighting the importance of measuring neutralising antibodies rather than total anti-RBD in assessing antibody-based immunity. The six healthy controls used in this experiment (H1-H6) showed no anti-RBD and no detectable inhibition. Example 13 - Neutralisation testing for variants of interest

[0264] In addition to waning levels of antibody over time, the long-term efficacy of antibody-based immunity to COVID-19 from vaccination or prior infection is being challenged by the emergence of variants of concern (VOC) for SARS-CoV-2, with single and multiple mutations in the spike protein and especially in RBD that may affect binding affinity to ACE2, transmissibility (RO) and/or susceptibility to neutralising antibodies. Testing of immunity may therefore require consideration of circulating VOCs. This assay was designed to facilitate the substitution of variant RBD proteins by the use of RBD-biotin and anti-biotin gold nanoparticles, four different versions of RBD-biotin were evaluated in our assay (Figure 28).

[0265] In this study, an N- and C-terminal truncated variants of the RBD sequence derived from the original Wuhan-Hu-1 strain (SEQ ID NO:3; residues 1332 to N532 of UniProtKB - PODTC2) and variants S477N, S477I and N439K were expressed with a C-terminal avi- tag (GLNDIFEAQKIEWHE), purified and titrated before mixing with a fixed amount of anti-biotin gold (Figure 28). For biotinylation, Expi293F cells were stably transfected with a plasmid directing expression of the BirA biotin ligase (ExpiBirA) for expression of the RBD-avi-tag constructs. For in situ biotinylation, media was supplemented with a final concentration of 50 micro molar D biotin and cells were incubated at 34°C after transfection. [0266] Each of the four RBD showed essentially identical titration curves for binding of RBD-biotin-Au complexes to ACE2 (Figure 28A-B). The susceptibility of each variant to inhibition by patient antibodies was assessed using longitudinal plasma samples from patient Cl (infected in February 2020) (Figure 28B-C). As shown in Figure 28B-C, the level of inhibition for all variants was around 90% at day 19 (Cl. 1) after PCR diagnosis, reducing to around 70-80% at 35 days (Cl.3) and around 40-60% at 72 days (Cl.5) for each of the Wuhan RBD and S477N, S477I, and N439K variant RBDs. These results show that the LFA can be easily modified to measure neutralising antibodies against variant RBD, with equivalent activity for this patient against these minor variant mutations, and would be expected to show differential sensitivity for neutralisation depending on the variant and the patient sample tested. As also shown in Figure 28C, the level of total antibody measured with each variant RBD-biotin-Au was similar at each time point. Example 14 - Measurement of neutralising antibodies in whole blood

[0267] Lateral flow assay devices may be suitably configured for use with whole blood samples, rather than with plasma or serum samples that otherwise require access to laboratory facilities and further sample processing prior to being applied to an LFA device. An LFA device for use with whole blood may employ a method of removing cells from the whole blood (e.g., red blood cells and / or white blood cells), in particular where the presence of such cells would interfere with assay performance. Suitable methods for configuring an LFA device to remove at least some of the cells from a whole blood sample applied thereto (red blood cells and / or white blood cells) will be familiar to persons skilled in the art, illustrative example of which include the use of differential filters that retain cells and allow plasma to flow through, and immunological approaches that use antibodies (e.g., anti- glycophorin A antibodies) or antigen-binding fragments thereof to agglutinate red blood cells (and optionally one or more types of white blood cells), suitably within an open-weave matrix, thereby allowing plasma (and optionally white blood cells) to flow through. In this context, immunological approaches are generally favoured, as methods based on physical filtration can sometimes retard the flow of plasma through the LFA device to a greater extent than methods based on blood cell agglutination. In the following examples, anti-glycophorin A agglutination was used to remove red blood cells, while allowing the free flow of plasma across the LFA device. However, it will be appreciated that other approaches could be applied to remove red blood cells and optionally white blood cells from a whole blood sample that is applied to the LFA device described herein.

[0268] To demonstrate that the use of whole blood is potentially compatible with the RBD- ACE2 inhibition assay, a sample of whole blood was spiked with varying concentrations of a neutralising anti-SARS-CoV-2 monoclonal antibody, mAb #42. The spiked samples were then analysed using the lateral flow assay devices as shown in Figure 27, but modified by (i) the addition of a 1:5 dilution of Epiclone™ monoclonal IgG anti-glycophorin A antibodies (Seqirus, Australia) to the sample application pad, and (ii) the use of a reduced amount of RBD-biotin (0.12 pg/ml versus 1 pg/ml). The modified LFA device is referred to as Version la. Spiked whole blood samples were added to Version la devices and the results were analysed using an Axxin AX-2X reader. The device strips were then removed from the cartridges for photography of the test strip, including the sample application pad to show retention of whole blood (see Figures 29A-C). [0269] To test whether the Version la device is compatible with clinical samples, serial plasma samples from subjects Cl and C2 (with known neutralising anti-SARS-CoV-2 antibody titres) were mixed with an equal volume of packed red blood cells to approximate the composition of whole blood, and analysed on the Version la device. As shown in Figure 29D, while inhibition is seen with each of the samples, only small differences were observed between the % inhibition across samples (from 27 to 69%) despite their wide range of ID50 titres (from 50 to 700).

[0270] A second version of the device (Version lb; Figure 30A) was assembled with the amount of RBD-biotin increased to the level of 1 pg/ml (as compared to 0.12 pg/ml in Version la of Figure 29). Whole blood, plasma or buffer (PBS pH 7.4) were spiked with 10 pg/ml of anti-SARS-CoV-2 neutralising antibody mAb #42 and assayed on the test strips. As shown in Figure 30B, even with very high concentrations of neutralising mAb, less inhibition is seen for plasma (81%) and for whole blood (55%) than for buffer (98%) in this version of the LFA device.

[0271] The Version la and Version lb LFA devices referred to above have a distance of more than 1 cm between the sample application pad and the conjugate pad containing RBD- biotin-gold complexes. As a consequence, the plasma within the samples (whether plasma or whole blood sample) is partially diluted by running buffer before it comes into contact with the RBD-biotin target antigen. To minimise this sample dilution effect, two further versions of the device were configured in which the RBD-biotin-gold conjugate was added to the sample application pad along with the anti-glycophorin A antibodies, so that the plasma of the sample immediately comes into contact with the RBD-biotin target antigen (see Figure 31). Versions lb, 2 and 3 were used to assay whole blood, plasma or buffer (PBS pH 7.4) spiked with 10 pg/ml of neutralising mAb #42. As shown in Figure 32, Versions 2 and 3 were more efficient at detecting neutralizing antibodies in spiked whole blood samples. [0272] Serial plasma samples from subjects Cl and C2 (with known neutralising antibody titres) were tested on Version 2 devices. The visual comparison of the test strips shown in Figures 33A and B, and the analysis depicted in Figure 33C show equivalence for whole blood versus plasma for each sample.

[0273] These data show that the LFA devices described herein may be suitably configured to detect the presence of neutralizing anti-SARS-CoV-2 antibodies in whole blood and provide good correlation between neutralising antibody content and % inhibition for both plasma and whole blood samples.

Example 15 - Detection of neutralizing antibodies that bind to regions outside of the RBD of SARS-CoV-2

[0274] While the RBD is the target antigen of around 90% of neutralising antibodies to SARS-CoV-2, there are epitopes outside of the RBD - including in the SI domain or full- length S protein - that may affect the tertiary structure of the RBD and subsequent binding of neutralising antibodies against the RBD. In addition, binding of antibodies to regions of the S protein that are adjacent to the RBD may block or otherwise inhibit binding of RBD to ACE2 through steric hindrance, even when the target epitope itself may he outside of the RBD. Thus, in some embodiments, there may be advantages to using the SI, full-length S or full-length S trimers or other fragments as the target antigen for ACE2 inhibition in the LFA devices described herein, so that the LFA can detect the effect of potential neutralising antibodies that bind to regions outside of the RBD and indirectly affect the interaction of RBD with ACE2.

[0275] To test this hypothesis, an LFA device was configured using, as the tracer antigen, the full-length trimeric form of the SARS-CoV-2 S protein (SEQ ID NO: 4, below; also referred to herein as FHA) directly conjugated to colloid gold (Au; DCNovations Colloidal Gold, DCN Dx, USA). The FHA construct was expressed with a C-terminal multi-His tag followed by an avi-tag (GLNDIFEAQKIEWHE; SEQ ID NO:5), as shown in SEQ ID NO:4, below, allowing for in vitro BirA biotin labelling, if and when required.

SEQ ID NO:4 (also showing the His tag (italicised) and the avi-tag (underlined))

VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVS GTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLD SKTQ SLLIVNNATNVVI KVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGK QGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDP LSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYA WNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEV RQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNL KPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFEL LHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADT TDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQ LTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPGSA SSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYI CGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFG GFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKF NGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIG VTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQ LSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN LAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTT APAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIV NNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEV AI<NLNESLIDLQELGI<YEQGSGSYIPEAPRDGQAYVRI<DGEWVLLSTFLGSGSW

WWWGSGSGLNDIFEAQKIEWHE

[0276] Plasma samples from two different patients with known SARS-CoV-2 neutralizing antibodies (Patient Cl at 3 different time points [Cl. l, C1.3 and C1.5], and Patient C2 at a single time point [C2. 1]) were run on the FHA device and, for comparison, on an LFA device employing colloid gold-labelled RBD as the tracer antigen. In these LFA devices, the RBD and the FHA antigens were conjugated directly to colloidal gold rather than using biotinylated antigens mixed with anti-biotin colloidal gold (BBI Solutions, UK), as described in the aforementioned Examples. This flexibility in methods for conjugation to gold or other detectable labels could be exploited to develop LFA devices with alternative detectable labels, illustrative examples of which include fluorescence, quantum dot and coloured latex.

[0277] As shown in Figure 34, the presence of neutralizing anti-SARS-CoV-2 antibodies in patient plasma inhibited FHA binding to the immobilized ACE2 capture antigen (B), with a similar pattern of inhibition to that seen with RBD (A). In both devices, there was robust binding of RBD and FHA to the immobilized ACE2 in the absence of neutralising anti- SARS-CoV-2 antibodies (negative (Neg) control). [0278] The quantitative data presented in Figure 35 show that for patient Cl, a higher level of inhibition in the FHA (full-length S) assay was observed at days 35 and 70 but not day 19 after onset when compared to the RBD assay, suggesting the presence of some neutralising antibodies directed against non-RBD epitopes in patient Cl that are developing over increasing time and/or decaying at a slower rate after day 19 than those directed at RBD epitopes. In contrast, for patient C2 at day 35, the level of inhibition observed was identical in both the RBD and the FHA assays, suggesting that the plasma sample from patient C2 had neutralising antibodies predominantly against RBD.

Example 16 - Detection of neutralizing antibodies in serum samples from macaques immunised with SARS-CoV-2

[0279] In this study, the LFA device described herein was used to assess the level of total and neutralizing anti-SARS-CoV-2 antibody titres in macaques immunised with an experimental SARS-CoV-2 spike vaccine that has been shown to induce a high titre neutralizing antibody response in macaques, as previously described in Tan etal. (2021, Nat Commun. 12: 1403). Briefly, 3 macaques were primed with spike protein vaccine in combination with Addavax™ adjuvant, and boosted with soluble RBD protein on day 21. Eight additional macaques were primed with whole SARS-CoV-2 spike protein (day 0) and boosted with spike protein (day 21), as described in Tan et al. (2021). Blood samples were taken prior to the prime (day 0), post-prime (day 14; five animals only), prior to the boost (day 21), and post-boost (day 42). As shown in Figures 36A and B, while some evidence of RBD-ACE2 inhibition was observed with the post-prime/pre-boost samples, the post-boost (day 42) samples all showed clear RBD-ACE2 inhibition ranging from 50% to 95%. The total anti-RBD line of the LFA test strip also became more prominent in all animals following the boost. These data demonstrate that the LFA device described herein can be used to detect the presence of total anti-RBD antibodies and anti-SARS-CoV-2 neutralising antibodies in samples from vaccine recipients. It is also noteworthy that these data demonstrate that the LFA device can be used to detect total and neutralizing antibodies in non-human derived samples and may therefore advantageously be used to assess SARS- CoV-2 immunity in animal models and in non-human species generally.

Claims

- 93 - CLAIMS:

1. A lateral flow assay device for detecting an anti-viral antibody in a biological fluid sample, the device comprising, in the direction of flow: a. a sample application region, wherein the sample application region is configured to receive a biological fluid sample; b. a conjugation region comprising a tracer antigen, wherein the tracer antigen comprises a detectable moiety; and c. a test region comprising an immobilised capture antigen; wherein the device is configured to move the biological fluid sample by capillary action in the direction of flow from the sample application region to the test region; wherein the tracer antigen or the immobilised capture antigen is a viral antigen or a functional variant thereof; and wherein

(ii) in the presence of an anti-viral antibody in the biological fluid sample, the antiviral antibody inhibits the formation of the complex comprising the tracer antigen and the capture antigen at the test region, thereby producing a weaker detectable test signal at the test region when compared to a reference signal that is representative of a test signal that is produced in absence of the anti-viral antibody.

2. The lateral flow assay device of claim 1, wherein the tracer antigen is a viral antigen or a functional variant thereof.

3. The lateral flow assay device of claim 2, wherein the viral antigen is of a virus selected from the group consisting of a picomavirus, a coronavirus, an influenza virus, a parainfluenza virus, a respiratory syncytial virus, an adenovirus, an enterovirus, and a metapneumovirus. - 94 -

4. The lateral flow assay device of claim 3, wherein the virus is a coronavirus.

5. The lateral flow assay device of claim 4, wherein the coronavirus is selected from the group consisting of MERS and SARS.

6. The lateral flow assay device of claim 5, wherein the coronavirus is SARS-CoV-2.

7. The lateral flow assay device of claim 6, wherein the viral antigen is a SARS-CoV- 2 spike protein.

8. The lateral flow assay device of claim 7, wherein the viral antigen comprises a receptor binding domain (RBD) of the SARS-CoV-2 spike protein.

9. The lateral flow assay device of any one of claims 2 to 8, wherein the immobilised capture antigen is a receptor to the viral antigen.

10. The lateral flow assay device of any one of claims 6 to 8, wherein the immobilised capture antigen is an angiotensin converting enzyme 2 (ACE2) or an RBD-binding fragment thereof.

11. The lateral flow assay device of claim 10, wherein the ACE2 is human ACE2.

12. The lateral flow assay device of claim 1, wherein the tracer antigen is a receptor to the viral antigen.

13. The lateral flow assay device of claim 12, wherein the receptor is an angiotensin converting enzyme 2 (ACE2) or an RBD-binding fragment thereof.

14. The lateral flow assay device of claim 13, wherein the ACE2 is human ACE2.

15. The lateral flow assay device of claim 13 or claim 14, wherein the immobilised capture antigen is a SARS-CoV-2 viral protein or a functional variant thereof.

16. The lateral flow assay device of claim 15, wherein the immobilised capture antigen is a receptor binding domain (RBD) of the SARS-CoV-2 spike protein.

17. The lateral flow assay device of any one of claims 1 to 16, wherein the detectable moiety is selected from the group consisting of colloid gold, biotin, streptavidin and any combination of the foregoing.

18. The lateral flow assay device of claim 17, wherein the detectable moiety comprises colloid gold. - 95 -

19. The lateral flow assay device of any one of claims 1 to 18, wherein the immobilised binding agent at the control region is capable of binding specifically to the tracer antigen.

20. The lateral flow assay device of any one of claims 1 to 18, wherein the immobilised binding agent at the control region is capable of binding specifically to the detectable moiety.

21. The lateral flow assay device of any one of claims 1 to 20, wherein the biological fluid is selected from the group consisting of blood, serum, plasma, saliva and nasopharyngeal secretion.

22. The lateral flow assay device of any one of claims 1 to 21, further comprising a control region comprising an immobilised control agent.

23. The lateral flow assay device of claim 22, wherein the immobilised control agent forms a complex with a labelled component of the fluid sample or with the tracer antigen to produce a detectable control signal at the control region.

24. The lateral flow assay device of claim 23, wherein the immobilised control agent forms a complex with the tracer antigen to produce a detectable control signal at the control region.

25. The lateral flow assay device of any one of claims 22 to 24, wherein the control region is downstream of the test region in the direction of flow.

26. The lateral flow assay device of any one of claims 22 to 25, wherein the control signal is representative of a test signal that is produced at the test region in absence of the anti-viral antibody.

27. The lateral flow assay device of claims 26, wherein the control signal has an intensity or value of from about 30% to about 70% of the intensity or value that is representative of the test signal produced at the test region in the absence of the anti-viral antibody.

28. The lateral flow assay device of any one of claims 22 to 25, wherein the control signal is representative of a test signal that is produced at the test region in the presence of the anti-viral antibody.

29. A method of detecting an anti-viral antibody in a biological fluid sample of a subject, the method comprising: - 96 -

(a) applying a biological fluid sample from a subject to the sample application region of the lateral flow assay device of any one of claims 1 to 28 for a period of time sufficient to allow the biological fluid sample and trace antigen to flow by capillary action to the test region;

30. The method of claim 29, wherein the reference test signal is representative of a detectable test signal produced by a biological fluid sample that does not contain the antiviral antibody.

31. A method of identifying a subject as being a source of neutralising anti-viral antibodies, the method comprising:

(a) obtaining a biological fluid sample from a subject;

(b) applying the biological fluid sample from step (a) to the sample application region of the lateral flow assay device of any one of claims 1 to 28 for a period of time sufficient to allow the biological fluid sample and trace antigen to flow by capillary action to the test region;

(c) comparing the detectable test signal at the test region with a reference test signal, wherein the subject is identified as a source of neutralising anti-viral antibodies when a weaker test signal is detected at the test line when compared to the reference test signal.

32. The method of claim 31, wherein the reference test signal is representative of a detectable test signal produced by a biological fluid sample that does not contain neutralising anti-viral antibodies.

33. The method of any one of claims 29 to 32, wherein the biological fluid is selected from the group consisting of blood, serum, plasma, saliva and nasopharyngeal secretion.

34. The method of any one of claims 29 to 33, wherein the subject is a human.

35. The method of any one of claims 29 to 33, wherein the subject is a non-human animal. - 97 -

36. A composition enriched for neutralising anti-viral antibodies obtained from the source identified by the method of any one of claims 31 to 35.

37. A method of treating or preventing viral infection in a subject in need thereof, the method comprising administering to the subject the composition of claim 36.

38. A method of identifying the presence of a neutralising anti-viral antibody in a sample, the method comprising:

(a) applying a sample to the sample application region of the lateral flow assay device of any one of claims 1 to 28 for a period of time sufficient to allow the sample and trace antigen to flow across the device to the test region;

(b) comparing the detectable test signal at the test region with a reference test signal, wherein the subject is identified as a source of neutralising anti-viral antibodies when a weaker test signal is detected at the test region when compared to the reference test signal.

39. The method of claim 38, wherein the reference test signal is representative of a detectable test signal produced at the test region in the absence of neutralising anti-viral antibodies.

40. The method claim 39, wherein the reference test signal has an intensity or value of from about 30% to about 70% of the intensity or value that is representative of the detectable test signal produced at the test region in the absence of neutralizing anti-viral antibodies.

41. The method of claim 38, wherein the reference test signal is representative of a detectable test signal produced at the test region in the presence of neutralising anti-viral antibodies.