WO2021186003A1

WO2021186003A1 - Improved lateral flow formats for optimized flow and increased sensitivity

Info

Publication number: WO2021186003A1
Application number: PCT/EP2021/057027
Authority: WO
Inventors: Robert Porter; Mohamad TAKWA; Vasiliki FRAGKOU; Milo Thomas WRIGHT
Original assignee: Aegirbio Ab
Priority date: 2020-03-18
Filing date: 2021-03-18
Publication date: 2021-09-23

Abstract

The present disclosure relates to a lateral flow device for detecting the presence of at least one target compound in a sample, the lateral flow device comprising: a membrane layer, comprising at least one test line, the at least one test line including an immobilized capturing agent for detecting the presence of the at least one compound in the sample; a reagent zone, comprising: i. a conjugate zone for receiving a running buffer, and ii. a sample loading zone for receiving the sample; wherein the sample loading zone is located, along the axial length of the lateral flow device, between the conjugate zone and the at least one test line.

Description

Improved lateral flow formats for optimized flow and increased sensitivity

Technical field

The present invention relates to lateral flow devices and methods for controlling the interactions between reagents and components of a sample.

Background

Lateral flow devices, or simply strip tests, are usually self-contained, portable devices that are easy to use, fast, and inexpensive. Lateral flow devices can be stored at ambient temperature, have a long shelf life, and provide diagnostic results without complex sample processing or additional equipment, making them ideal for both point- of-care and field-based diagnostic uses.

The purpose of these devices are to detect a target compound in a liquid sample, where the detection can be done both semi-quantitatively or quantitatively. For some lateral flow devices it is not necessary to quantitate the target compound, it is enough to detect or not detect the presence of a target substance, such as pregnancy tests or screening for microbial infections.

A typical lateral flow device is composed of several porous materials, onto which assay reagents are striped, sprayed, or spotted, then dried for storage in spatially distinct locations, and through which analyte, in a clinical or environmental sample, and assay reagents are transported by capillary action. Commonly, a sample pad is used for receiving a sample, localized at one end of the strip. The sample will then flow by capillary action through a conjugate pad, where conjugates, comprising indicator particles (such as colloidal gold) together with detection/capture molecules specific for the target compound, have been deposited and dried. When the sample gets in contact with the dried conjugates, they dissolve and bind to the target compound in the sample, thereby forming a complex. This complex continues to flow through the membrane of the lateral flow device, where there are one or more test lines for the target compounds, The test lines contain target-specific molecules, e.g. antibodies. For a sandwich assay, a visually present test line indicates that the target compound has bound to the test line, and that it therefore is present in the sample. For a competitive assay, it is instead the absence of a visually present test line that indicates the presence of the target compound in the sample. Additionally, the lateral flow device typically comprises a control line, which binds and immobilizes unbound conjugates. A visually present control line indicates that the test has operated correctly.

While lateral flow devices have gained widespread use, other methods, for example other antibody-based tests such as ELISA, are typically associated with a superior sensitivity compared to conventional lateral flow devices. Several factors have been considered in order to increase the sensitivity of lateral flow devices, including the transport dynamics, the reaction kinetics, the signal generation and the subsequent detection of the output signal. However, the sensitivity of the lateral flow device is still inferior to comparable analytical instruments, limiting the use of lateral flow devices.

Hence, there is a need for novel approaches to lateral flow devices, which has the possibility to overcome these disadvantages and thus exhibiting higher sensitivity and specificity in order to open up for the use of lateral flow devices to other types of applications, wherein a higher sensitivity and specificity is required.

Summary

The present inventors have realized that although the interactions between the sample and the reagents, such as the conjugates, are an essential component in the generation of a visible signal, they can also have a detrimental effect to the sensitivity and specificity of lateral flow devices. Therefore, by controlling the interactions the negative effects can be mitigated.

The conventional arrangement, wherein conjugates are deposited on a conjugate pad and allowed to interact with the sample upon dissolution, may not result in optimal interactions between the sample and the conjugates. For example, although a target compound is present in the sample, a substantial amount of conjugates may be dissolved and arrive at the test line without having been given the opportunity to interact with the target compound, potentially leading to inconclusive test results.

Furthermore, interactions between the sample and conjugates may lead to aggregation, and the formation of large complexes that are not able to reach the test lines. The agglutinate may further act to restrict fluid flow of the device and alter the displacement of the sample and/or conjugates. A network of conjugates and target compounds may be formed for example if the target compound comprises multiple copies of the epitope towards which the conjugate is directed. Additionally, for a competitive assay, if the conjugate reaches the target line before the sample, the sensitivity and specificity of the test decreases.

Therefore, in a first aspect, the invention relates to a lateral flow device for detecting the presence of at least one target compound in a sample, the lateral flow device comprising:

I. a membrane layer, comprising at least one test line, the at least one test line including an immobilized capturing agent for capturing the at least one compound in the sample;

II. a reagent zone, comprising:

• a conjugate zone for receiving a running buffer, and

• a sample loading zone for receiving the sample; wherein the sample loading zone is located, along the axial length of the lateral flow device, between the conjugate zone and the at least one test line.

While the analysis of a sample, comprising a target compound having multiple copies of an epitope, by conventional lateral flow devices may lead to formation of large complexes. The present disclosure may prevent this from occurring. Thereby, the fluid flow through the device may not be restricted by large complexes which may act to exclude at least a part of the sample and/or the conjugates from reaching the test lines and/or control lines. An important aspect of the present disclosure is therefore mitigation of the drawbacks associated with complex formation, at undesirable locations of the lateral flow device, which could result in non-optimal flow behaviour and, as a result, a decreased sensitivity and specificity.

The location of the sample loading zone, with respect to the conjugate zone and the test lines, has been revealed to be an important factor for achieving lateral flow devices with an optimal flow behaviour. By controlling the interactions between the conjugates and the sample, such as the target compound, the sample and the conjugates can avoid forming complexes which may result in a substantial fraction of the sample and/or the conjugates not reaching the test line or the control line. The lateral flow device may comprise conjugates, typically provided in a dry state as part of the conjugate zone. Additionally, or alternatively, the conjugates may be provided as part of a buffer solution and/or a running buffer. In such a case, the running buffer and/or buffer solution may also be referred to as a conjugate solution. Typically the conjugates comprise a label and at least one conjugate protein binders. The conjugates may thereby be configured to bind to the one or more target compounds and, thereby provide an indication of the presence of the target compound in the sample.

Depending on the configuration of the lateral flow device, the indicator for the presence of the target compound may be a visually observable line at the test line, or the absence of a visually observable line at the test line, preferably in combination with a visually observable line at the control line. Typically a sandwich assay generates a visually observable line, if the target compound is present in the sample, while a competitive assay would result in no visually observable line at the test line. However, the lateral flow device may be configured with a combination of a number of sandwich assay test lines and a number of competitive test lines. In general, it may be a preference that the control line is visually present for indicating that the test has operated correctly

The conjugate zone is typically located near an end of the lateral flow device, such as a proximate end. By this arrangement, the proximate end may be put in contact with a buffer solution, potentially comprising the conjugates. The solution will thereafter, by capillary action, flow through the device, and may act to drive the sample towards the test line, while maintaining a control of the interactions between the sample and the conjugates. Preferably, the sample reaches the test line before the conjugates, thereby the capturing agent may bind to the target compound, if provided in a sandwich assay format, and the conjugates may thereafter bind and form a visually observable indication of the presence of the target compound.

In an embodiment of the present disclosure, the lateral flow device comprises a filter for filtering a sample before contacting the membrane layer. The filter may be provided to cover at least a part of the sample loading zone. The filter may be a porous structure with pores in the size range between 0.1 and 1 pm. The filter may for example be configured to filter out mucus structures of the sample. In a further aspect, the invention relates to a kit for detecting the presence of a target compound in a sample, the kit comprising:

• a running buffer; and

• a lateral flow device comprising: o a membrane layer, comprising at least one test line, the at least one test line comprising an immobilized capturing agent for capturing the at least one target compound in the sample; and o a reagent zone comprising:

^■ a conjugate zone for receiving the running buffer; and

^■ a sample loading zone for receiving the sample; wherein the sample loading zone is located, along the axial length of the lateral flow device, between the conjugate zone and the at least one test line.

In a preferred embodiment of the present disclosure, the kit further comprises a buffer solution for mixing with the sample. The buffer solution may be selected to decrease non-specific binding when performing the test, dissolve components, such as mucus, of the sample, stabilizing the sample, and/or to adjust the viscosity of the sample.

The kit may be provided with a receptacle containing the buffer solution. Preferably, the sample is to be mixed with the buffer solution at a ratio of between 1:5 and 1:15, such as at a ratio between 1:7 and 1:12. It is a preference that the sample is a saliva sample. Saliva samples have been shown to comprise numerous relevant disease markers, and the process of obtaining saliva samples is associated with limited inconvenience for the patient, as compared to collection of other sample types, such as a nasopharyngeal sample. For example 0.1-1 ml of saliva may rapidly be collected from the mouth of a patient and has been shown to allow for sensitive quantitative and/or qualitative measurements of a target analyte in a sample.

Depending on the configuration of the lateral flow device, the kit may advantageously be provided, in the dry unused state, with a conjugate zone comprising conjugates. The conjugates may for example have been striped, sprayed, or spotted, then dried on the conjugate zone before use of the lateral flow device. When in use, a fluid, such as the running buffer, may act to displace the conjugates towards the one or more test lines, a control line and/or an absorption pad. Additionally or alternatively, the conjugates may be provided as part of the running buffer. The kit may for example comprise a receptacle retaining the running buffer, and may further be configured such that the running buffer/conjugate solution is applied to the lateral flow device by exposing the proximate end of the lateral flow device to the running buffer/conjugate solution. The fluid may act to transport the sample to the test line without the formation of a significant amount of complexes comprising the conjugate and the target compound at undesirable locations.

In yet a further aspect, the invention relates to a method for detecting the presence of at least one target compound in a sample, the method comprising the following steps:

I. providing a kit comprising:

• a running buffer; and

• a lateral flow device comprising:

- a membrane layer comprising at least one test line; and

- a reagent zone comprising:

• a conjugate zone for receiving the running buffer; and

II. applying the sample to the sample loading zone;

III. applying the running buffer to the conjugate zone;

IV. waiting a sufficient amount of time for the sample and the conjugates to arrive at the test lines; and

V. detecting, for each test line, the presence of at least one indicator; thereby detecting the presence of at least one target compound in the sample.

As discussed above, depending on the assay type, indicators may either be the presence of a visible line or the absence of a visible line. For example if the test line is a sandwich assay or a competitive assay. The lateral flow device may further be configured with a combination of one or more sandwich assays and one or more competitive assays. Due to the high sensitivity, a wide range of target compounds can be detected, even at low concentrations. For example, the target compound may be a biomarker for a viral disease, such as coronavirus infection. The capturing agent, at the test line, may in such a case comprise a synthetic fragment of the virus, such as an epitope. In this arrangement, the epitope of the test line, may be substantially identical, such as functionally identical, to the binding counterpart of the conjugate.

It may be a preference to wait a predetermined time, between the step of applying the sample and the step of applying the running buffer. For example in order to allow the sample to reach the test line before the application of conjugates. Alternatively it should be appreciated that the running buffer may be added to substantially the same location of the lateral flow device as the sample.

Description of Drawings

Fig. 1 shows a schematic illustration and the use of a lateral flow device, according to a specific embodiment of the present disclosure.

Fig. 2 shows a schematic illustration and the use of a lateral flow device comprising a conjugate zone that, before use, had been preloaded with conjugates, according to a specific embodiment of the present disclosure.

Fig. 3 shows schematic illustrations of a lateral flow device comprising three test lines and a control line, and its use, according to a specific embodiment of the present disclosure..

Fig. 4 shows schematic illustrations of possible outcome of measurements of patient blood by a lateral flow device for viral infections, according to a specific embodiment of the present disclosure.

Fig. 5 shows lateral flow devices following measurements of IgG, IgM and virus particles, according to a specific embodiment of the present disclosure..

Fig. 6 shows a schematic illustration of a lateral flow device for serological assay IgG/lgM, according to a specific embodiment of the present disclosure.. Fig. 7 shows a schematic illustration of a lateral flow device for detection of virus particles, according to a specific embodiment of the present disclosure..

Fig. 8 shows lateral flow devices following measurements of virus particles, according to a specific embodiment of the present disclosure.

Fig. 9 shows a schematic illustration of a lateral flow device according to a specific embodiment of the present disclosure.

Fig. 10 shows a schematic illustration of a lateral flow device according to a specific embodiment of the present disclosure.

Detailed description

In a first aspect, the present invention relates to a lateral flow device for detecting the presence of a target compound in a sample. The device may comprise a membrane layer and a loading zone. Said loading zone may be provided as a part of the membrane layer or may be a separate component, but preferably in fluidic contact with the membrane layer. The membrane layer may comprise one or more test lines comprising immobilized capturing agent(s) specific for one or more target compounds. Typically, each test line comprises a specific type of capturing agent. Thereby, each test line is directed towards a specific target compound. Furthermore, the lateral flow device may comprise a loading zone. Said loading zone may comprise a conjugate zone and a sample loading zone, wherein the conjugate zone may comprise the conjugates and/or wherein the conjugate zone is configured to receive the conjugates, typically after the sample/sample solution has been provided to the sample loading zone. The sample loading zone and the conjugate zone may be provided as part of a single unit, for example a reagent zone or a reagent pad. However, the sample loading zone and the conjugate zone may further be provided as separate units, preferably still in fluidic contact. It is a preference that the sample loading zone is located, along the axial length of the lateral flow device, between the conjugate zone and the at least one test line. Typically, the lateral flow device comprises an absorption pad at a distal end of the lateral flow device. It is a preference that the proximate end of the device comprises the conjugate zone. It is a preference that the buffer solution is applied to the device by dipping the proximate end of the device into said buffer solution. Consequently, it may be a preference that the conjugate zone is located at, or near, the proximate end of the device. In general, it is a preference that the conjugate zone is located towards the proximate end of the device with respect to the sample loading zone.

In an preferred embodiment of the present disclosure, the sample loading zone is located between the conjugate zone and the test line, thereby allowing the sample to flow before the conjugate, which will decrease the risk of the sample and conjugate mixing and potentially aggregating, thereby increasing the risk of them not reaching the test lines.

As used herein "conjugate" or "conjugated" refers to two molecules that are bound to each other. For example, it may refer to a particle, such as gold, latex or any other colloidal material, bound to the target compound or a protein containing the epitope or epitopes of the target compound. The particle can also be bound to other detection/capture molecules specific for the target compound.

As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly states otherwise. Thus, for example, reference to “an antibody” includes a plurality of such antibodies.

The term "aptamer" as used herein refers to a single-stranded oligonucleotide (single- stranded DNA or RNA molecule) that can bind specifically to its target with high affinity. The aptamer can be used as a biosensor element capable of binding to a molecule in a detection/analysis system, and thus has been recognized as a substitutive for antibody. Particularly, the aptamers can be used as molecules targeting various organic and inorganic materials, including toxins, unlike antibodies, and once an aptamer binding specifically to a certain material is isolated, it can be consistently reproduced at low costs using automated oligomer synthesis methods. Further, the term “affimer” refers to small, engineered non-antibody binding proteins that can bind target molecules and are designed to mimic the molecular recognition characteristics of monoclonal antibodies. Affimer reagents are suitable for use in for example biosensors and point-of-care diagnostics. As used herein "polymer" refers to macromolecular materials having at least five repeating monomeric units, which may or may not be the same. The term polymer, as used herein, encompasses homopolymers and copolymers. A molecularly imprinted polymer is a polymer that has been processed using the molecular imprinting technique which leaves cavities in the polymer matrix with an affinity for a chosen template molecule. Molecularly imprinted polymers possess the most important features of biological receptors - recognition. Molecularly imprinted polymers can comprise cross linked polymers. They can also comprise amorphous metal oxides or zeolites. Metal oxides and zeolites can be imprinted using a variety of known techniques. In some cases, the cavities or pores produced are an induced fit for polymers of the imprinting molecules.

In one embodiment of the present disclosure, the conjugate zone of said lateral flow device comprises conjugates. For example, the conjugate zone, in the dry unused state, may comprise at least one type of conjugate. The conjugates may thereby have been preloaded on the device, such as on a conjugate pad. Conjugates may for example be applied to the conjugate zone with the use of an air jet dispensing platform or by immersion. Several machines with hollow fibre dispensers used to stripe nitrocellulose membranes can also be configured with an air jet spray apparatus to dispense the conjugates onto the conjugate zone.

In a further embodiment of the present disclosure, the conjugate zone does not comprise conjugates.

In an embodiment of the present disclosure a conjugate comprises a label and at least one conjugate protein binder, such as multiple conjugate protein binders. The conjugate protein binders may be configured for binding to an immobilized capturing agent, at any of the test lines. In such cases, the presence of a target compound at a test line, may result in the conjugate not being able to bind to the immobilized capturing agent. The conjugate protein binder may further be configured to bind to a target compound. In such cases the absence of a target compound at a test line may result in the conjugate not being immobilized at the test line. The conjugate protein binder, in combination with the capturing agent and the target compound, may be configured for binding according to a sandwich assay or a competitive assay. It should be appreciated that a lateral flow device comprising multiple lines, such as test lines and control lines, may comprise lines configured for sandwich assay format and lines configured for competitive assay format.

In a further embodiment of the present disclosure, the label is a metal nanoparticle, such as a gold nanoparticle, and/or a coloured latex particle. A person skilled in the art will appreciate that the capturing agent may further be any other type of sol-based material. A sol is a colloid made out of very small solid particles in a continuous liquid medium. Colloidal gold is the most widely used label in lateral flow devices, it has an intense colour and no development process is needed for visualization. Moreover, it has high stability in both liquid and dried forms. Another popular label is latex, which can be tagged with a variety of detector reagents such as coloured or fluorescent dyes, and magnetic or paramagnetic components. As latex can be produced in multiple colours, it has an application in multiplex assays, which require discrimination between numerous lines. Carbon, such as carbon nanotube, fluorescent labels, or enzymatic modification of the labels, may also be used to improve the sensitivity of the assay. The label may further be a fluorescent nanoparticle, such as a quantum dot.

In a further embodiment of the present disclosure, the conjugate protein binder is selected from the list including an antibody, an Ig unit, an aptamer, an affimer, an RNA molecule, a DNA molecule, an organic polymer, or a fragment thereof. The conjugate may comprise a single or multiple conjugate protein binders. In yet a further embodiment of the present disclosure, the organic polymer is a molecularly imprinted polymer.

In another embodiment of the present disclosure, the membrane layer of said lateral flow device comprises multiple test lines for capturing and/or detecting the presence of multiple target compounds in the sample. A multiplexed lateral flow device preferably comprises multiple test lines, wherein each test line comprises a binding agent for a specific target compound. The lateral flow device may thereby for example detect the presence of pathogens, such as virus or bacteria, while simultaneously having test lines comprising binding agents directed at capturing and/or detecting the presence of IgG and IgM in the sample. The presence of IgG and IgM should here be understood as an elevated concentration of IgG and IgM, as to normal reference values, such as reference values for the patient group of the individual which is diagnosed. This is important as when a patient undergoes an infection the pathogen, such as a virus, goes through a stage of replication and will elicit an immune response. In a first stage, the immune system produces IgM antibodies. Once the infection is under control, the immune system commences producing a more effective and long-term IgG response in order to eradicate the infection and prevent a future re-infection.

Thereby, in general a positive IgM test indicates that the subject has been infected and that the subject’s immune system has started responding to the pathogen. When IgM is detected the subject may still be infected, or the subject may have recently recovered from the pathogen infection.

In most subjects IgG is developed within seven to ten days after manifestation of symptoms of the pathogen infection has begun, and the IgG antibodies remain in the blood after an infection has passed. Consequently, IgG antibodies indicate that the subject has had a viral infection in the recent past and has developed antibodies that may protect the subject from future infection.

In order to perform a clinical staging of the subject, i.e. classify the subject by the infection stage, the presence of IgG, the presence of IgM and the presence of pathogens, such as bacteria and/or virus may be used. The clinical staging may result in six different scenarios as shown in the table below.

Table 1. Patient triage. A test strip that is capable of measuring all three parameters, according to table 1, will aid in the triage and allows for a rapid decision of a need for medical intervention. Clinical symptoms of an infection, such as COVID-19, may be taken into account during triage. For example, if a measurement by the lateral flow test strip reveals that the subject has developed IgG antibodies but no IgM or pathogens are detected, the clinical symptoms may be used to classify whether the subject is coming to an end of the infection, or whether the immune system is not capable of eliminating the pathogen, and thereby requires urgent medical intervention.

In yet another embodiment of the present disclosure each test line comprises a different immobilized capturing agent, such as a first capturing agent, a second capturing agent, a third capturing agent and/or a fourth capturing agent. In a specific embodiment of the present disclosure, the lateral flow device has a test line comprising a first capturing agent, another test line comprising a second capturing agent and yet another test line comprising a third capturing agent. In specific examples, the lateral flow device may further comprise even yet a further test line comprising a fourth capturing agent. Typically, the first capturing agent may be selected to capture IgM antibodies, the second capturing agent may be configured for capturing IgG antibodies, the third capturing agent may be configured for capturing pathogen particles, complexes thereof or fragments thereof. The fourth line may be a control line configured to provide a response, typically a visual response, of an accurate measurement.

In a further embodiment of the present disclosure each test line is configured such that it can detect the presence of a different target compound.

In one embodiment of the present disclosure, the lateral flow device is configured for operation as a sandwich assay. A person skilled in the art will appreciate that sandwich assays generally are used for larger compounds since they tend to have multiple binding sites. In a sandwich assay, the sample will come in contact with the conjugate and migrate together towards the test line of the lateral flow device, where a visually present test line indicates the presence of the target compound. The sandwich assay format is typically used for detecting larger analytes that have at least two binding sites, or epitopes. Usually, an antibody to one binding site is conjugated to the nanoparticle, and an antibody to another binding site is used for the assay’s test line. If there is analyte present in the sample, the analyte will bind to both the antibody-nanoparticle conjugate and to the antibody on the test line, yielding a positive signal. The sandwich format results in a signal intensity at the test line that is directly proportional to the amount of analyte present in the sample. Regardless of the quantity of analyte in the sample, an anti-species antibody at the control line will bind the nanoparticle, yielding a strong control line signal that demonstrates that the assay is functioning correctly.

The competitive format is typically used for detecting analytes when antibody pairs are unavailable or if the analyte is too small for multiple antibody binding events, such as steroids and drugs. In this format, the test line typically contains the analyte molecule, usually a protein-analyte complex, and the conjugate pad contains the detection antibody-nanoparticle conjugate. If the target analyte is present, the analyte will bind to the conjugate and prevent it from binding to the analyte at the test line. If the analyte is not present, the conjugates will bind to the analyte at the test line, yielding a signal. In the competitive format, the signal intensity is inversely proportional to the amount of analyte present in the sample. As in the sandwich format, the control line will bind the nanoparticle conjugate with or without the analyte providing confidence that the assay is working correctly. Typically, the conjugate can be said to be a detection particle, as it may, in certain embodiments of the present disclosure, provide an observable signal at a test line regarding the presence of a specific particle, such as a pathogen, in the sample.

In another embodiment of the present disclosure, the lateral flow device is configured for operation as a competitive assay. A person skilled in the art will appreciate that competitive assays generally are used for smaller compounds, but is also preferable for compounds with multiple epitopes.

In another embodiment of the present disclosure, the lateral flow device simultaneously functions as both a sandwich assay and a competitive assay. In another embodiment of the present disclosure, the membrane layer of said test directly contacts the test line. The immobilized binding agent may thereby be immobilized to a part of the membrane layer.

In one embodiment of the present disclosure, the conjugate zone of said test is located at a proximate end of the lateral flow device, such as wherein the test line is towards the distal end of the lateral flow device with respect to said conjugate zone.

In another embodiment of the present disclosure, the lateral flow device further comprises an absorbent pad, located at a distal end of the lateral flow device. The absorbent pad may for example be provided as part of the reagent zone. An absorbent pad allows for controlled release of sample and conjugate onto the lateral flow device and to filter undesired components. The absorbent pad may be in any type of material that absorbs water, such as cellulose fibre or woven meshes. Preferably the application pad comprises the conjugate zone and/or the sample loading zone, such as a conjugate pad and/or a sample pad. In a particular embodiment of the present disclosure the membrane layer comprises the reagent zone.

The absorbent pad may be configured to increase the total volume of sample that can enter the test strip. The bed volume of any membrane is finite, and having an absorbent pad at the distal end of the test strip can increase the volume of sample that can be run across the membrane as it acts as a sponge for the additional volume. As such, the presence of an absorbent pad can contribute to the reduction of non-specific binding and sensitivity. This may be accomplished due to the additional volume that can run across the test line washing non-specifically bound material off the test line, and allowing for an increase in totally analyte concentration to reach the test line.

In yet another embodiment of the present disclosure, the lateral flow device further comprises an application pad, and wherein the application pad comprises the conjugate zone and/or the sample loading zone.

In one embodiment of the present disclosure, the lateral flow device comprises a backing layer in an inert material. Inert materials are materials which have minimal or almost no chemical or biological activity when present in the environment. It is a strong preference that the membrane layer comprises or consists of a porous layer for wicking of a liquid sample. In an embodiment of the present disclosure, the membrane layer of said test comprises or consists of nitrocellulose, cellulose acetate or paper.

In a further embodiment of the present disclosure, the lateral flow device comprises a cassette, and/or wherein the lateral flow device is configured such that is can be operated as a dipstick

In one embodiment of the present disclosure, the capturing agent, such as any of a first, second, third or fourth capturing agent, of said lateral flow device comprises a capture protein binder. In another embodiment of the present disclosure, said capture protein binder is selected from the list including an antibody, an aptamer, an affimer, an RNA molecule, a DNA molecule, an organic polymer, the target compound, a derivative of the target compound, or a fragment thereof. The capture protein binder may for example comprise a virus particle, or a fragment thereof, for example attached to a colloidal particle at one or more of the test lines. In yet another embodiment of the present disclosure, said organic polymer is a molecularly imprinted polymer.

As disclosed elsewhere herein, the lateral flow test typically comprises multiple test lines, where each test line is configured to capture and/or detect a different compound. In cases wherein the lateral flow device comprises multiple test lines, it is a preference that the device comprises one test line configured for capturing IgM, one test line configured for capturing IgG, one test line configured for capturing a pathogen particle, such as a virus particle. Further, the lateral flow device typically also comprises a test line for providing an indication of an accurate measurement.

In an embodiment of the present disclosure, any of the capturing agents, such as any of a first, second, third or fourth capturing agent, comprises a capture protein binder, typically wherein each capturing agent has a unique capture protein binder. The capture protein binder may be selected from the list including an antibody, an aptamer, an affimer, an RNA molecule, a DNA molecule, an organic polymer, the target compound, a derivative of the target compound or a fragment thereof. The capture protein binder may for example comprise a virus particle, or a fragment thereof, for example attached to a colloidal particle at one or more of the test lines. In yet another embodiment of the present disclosure, said organic polymer is a molecularly imprinted polymer. In one embodiment of the present disclosure, the capturing agent and/or conjugate of the lateral flow device disclosed herein, is specific for an epitope of the target compound. In another embodiment of the present disclosure, said target compound comprises multiple copies of the epitope. In one embodiment of the present disclosure, the membrane layer of the lateral flow device disclosed herein, comprises a control line. In another embodiment of the present disclosure, said control line comprises an immobilized capturing agent specific for binding to the conjugate. In yet another embodiment of the present disclosure, said control line comprises the target compound, or a fragment thereof.

In a preferred embodiment of the present disclosure, the lateral flow device comprising a reagent zone (2) and a membrane layer (3). The test lines are here shown as visually observable and comprises a first test line (11), a second test line (12), and a third test line (13). The device furthermore comprises a control line (14). In a specific embodiment of the present disclosure the lateral flow device is targeting triaging of viral infections. For the three test line the first test line is configured to detect the presence of virus in the sample, thereby comprising a capturing agent specific for an epitope of the surface of the virus particle (19), the second test line is configured to detect the presence of IgM, the capturing agent may consequently be an anti-human IgM antibody (20), the third test line is configured to detect the presence of IgG, the capturing agent may consequently be anti-human IgG antibody (21). The control line is configured to target a conjugate, such as a non related control line conjugate, and may consequently be an antibody targeting this conjugate (22). Kit

In another aspect, the invention relates to a kit for detecting the presence of a target compound in a sample. The kit may comprise a lateral flow device and reagents. For example the kit may comprise a running buffer, for example that acts as a chase buffer, the kit may alternatively or additionally comprise a buffer solution to be mixed with the sample thereby forming a sample solution that is to be added to the lateral flow device. The buffer solution may for example act to stabilize and/or modify the viscosity of the sample. Furthermore, the kit may comprise a lateral flow device comprising a membrane layer. The membrane layer may comprise at least one test line comprising an immobilized capturing agent. Furthermore, the lateral flow device may comprise a reagent zone, such as for receiving sample, conjugates, and/or buffer solution. The reagent zone may comprise a conjugate zone for receiving the running buffer, and/or a sample loading zone for receiving the sample. It is a preference that the sample loading zone is located, along the axial length of the lateral flow device, between the conjugate zone and the at least one test line. Typically the lateral flow device comprises an absorption pad at a distal end of the lateral flow device. Near the opposite end, the proximate end, of the strip, it may be advantageous to have the conjugate zone located. Thereby, the running buffer may be applied to the conjugate zone by dipping the proximate end of the lateral flow device in the buffer solution. It is a preference that the conjugate zone is nearer the proximate end than the sample loading zone.

In one embodiment of the present disclosure, the kit comprises a buffer solution, the buffer solution comprising sodium chloride, bovine serum albumin, and a borate buffer. In one embodiment of the present disclosure, the kit comprises a buffer solution, the buffer solution consisting of sodium chloride, bovine serum albumin, and a borate buffer.

In one embodiment of the present disclosure, the kit comprises a buffer solution, the buffer solution comprising 1 M sodium chloride, 1% w/v bovine serum albumin, and 0.1M borate buffer.

In one embodiment of the present disclosure, the kit comprises a buffer solution, the buffer solution consisting of 1 M sodium chloride, 1% w/v bovine serum albumin, and 0.1M borate buffer.

In one embodiment of the present disclosure, the kit comprises a lateral flow device configured as disclosed elsewhere herein.

In one embodiment of the present disclosure, the conjugate zone of said kit comprises, in the dry unused state, the conjugates. In another embodiment of the present disclosure, said conjugates are provided in a dry state, and upon application of a mobile phase, said conjugates will flow substantially with the mobile phase.

In yet another embodiment of the present disclosure, said kit comprises a receptacle comprising the conjugates, as part of a conjugate solution. The lateral flow device is lowered into the conjugate solution in a conjugation well without lowering the sample loading zone in the buffer. The conjugates in the conjugate solution will flow into the membrane and continue along the lateral flow device.

In yet another embodiment of the present disclosure, the conjugates are configured such that upon application of the running buffer, said conjugates will flow substantially with the running buffer, such as from a proximate end of the lateral flow device to a distal end of the lateral flow device.

In yet another embodiment of the present disclosure, the conjugates are provided as part of the running buffer, or such that the conjugates can be mixed with the running buffer before application to the lateral flow device.

In yet another embodiment of the present disclosure, the kit comprises a receptacle for retaining the running buffer.

In an embodiment of the present disclosure, the kit is configured such that the running buffer may be applied to an end of the lateral flow device, such as the proximate end, by pipetting, and/or such that the running buffer may be applied to the lateral flow device by dipping an end of said lateral flow device, such as the proximate end, in the receptacle.

Method

In one aspect, the invention relates to a method for detecting the presence of at least one target compound in a sample. The method may comprise the provision of a kit as disclosed herein. The method may comprise a lateral flow device and reagents. For example the kit may comprise a running buffer and/or a buffer solution. Furthermore, the method may comprise the provision of a lateral flow device comprising a membrane layer. The membrane layer may comprise at least one test line comprising an immobilized capturing agent. Furthermore, the lateral flow device may comprise a reagent zone, such as for receiving sample, conjugates, and/or buffer solution. The reagent zone may comprise a conjugate zone for receiving the running buffer, and/or a sample loading zone for receiving the sample. It is a preference that the sample loading zone is located, along the axial length of the lateral flow device, between the conjugate zone and the at least one test line. Typically the lateral flow device comprises an absorption pad at a distal end of the lateral flow device. Near the opposite end, the proximate end, of the strip, it may be advantageous to have the conjugate zone located. Thereby, the running buffer may be applied to the conjugate zone by dipping the proximate end of the lateral flow device in the buffer solution. It is a preference that the conjugate zone is nearer the proximate end than the sample loading zone. The method may further comprise the step of applying the sample to the sample loading zone. Preferably the lateral flow device, and the sample volume, is configured and/or selected such that the majority of the sample progress towards the absorption pad, and does not interact with any present conjugates. Furthermore, the method may comprise the step of applying the running buffer to the conjugate zone. Thereby, the running buffer may reconstitute the conjugates, if provided preloaded on the lateral flow device. Further the method may comprise the step of waiting a sufficient amount of time for the sample and the conjugates to flow over the test lines. The sufficient time may for example be until the control line is visually observable. Furthermore, the method may comprise the step of detecting, for each test line, the presence of at least one indicator. The method may thereby be used for detecting the presence of at least one target compound in the sample. The method may also comprise the step of mixing the sample with the buffer solution, wherein the volumetric ratio between the buffer solution and the sample is between 5 parts buffer solution and 1 part sample and 15 part buffer solution and 1 part sample, thereby obtaining a sample solution. This step may be important for reducing the viscosity of the sample and allowing a sensitive and specific detection of the at least one target compound. Further, the method may comprise the step of applying the sample solution to the sample loading zone. Further the method may comprise the step of waiting a sufficient amount of time for the sample and the conjugates to flow over the test lines. The sufficient time may for example be until the control line is visually observable. Furthermore, the method may comprise the step of measuring, such as detecting, for each test line, the presence of at least one indicator. The method may thereby be used for detecting the presence of at least one target compound in the sample. In a specific embodiment of the present disclosure, the kit is configured as disclosed elsewhere herein, and/or wherein the lateral flow device is configured as disclosed elsewhere herein.

In a preferred embodiment of the present disclosure, the application of the sample and/or the application of the running buffer is configured such that a substantial fraction of the sample is allowed to flow over the test line before the conjugates.

In one embodiment of the present disclosure, the sample is first allowed to flow over the test lines before loading the conjugate. A person skilled in the art will appreciate that this prevents the conjugate and sample from mixing prior to reaching the test line, thereby avoiding aggregation on the test lines.

Method 2 - conjugates on lateral flow device

In one embodiment of the present disclosure, the lateral flow device of said method is provided with the conjugates present at the conjugate zone, such as in the dry unused state.

In another embodiment of the present disclosure, the running buffer of said method does not comprise conjugates.

Method 1 - conjugates in solution

In one embodiment of the present disclosure, the lateral flow device of said method is provided without the conjugates present at the conjugate zone.

In another embodiment of the present disclosure, the running buffer of said method comprises conjugates

Sample/conjugate addition and readout

In one embodiment of the present disclosure, the running buffer of said method is added to the loading zone, such as the conjugate zone, and/or a proximate end of the lateral flow device, at or near the conjugate zone.

In an embodiment of the present disclosure, the running buffer is first applied to an area between the sample loading zone and the conjugate zone, followed by a second application of running buffer to the conjugate zone and/or the proximate end of the lateral flow device, wherein the first application is carried out for a predetermined time and/or consists of application of a predetermined volume.

In another embodiment of the present disclosure, said running buffer is applied to the loading zone by dipping an end of the lateral flow device in the running buffer.

In yet another embodiment of the present disclosure, the sufficient time is the time lapsed until the control line is expected to be detectable, such as visually detectable.

In one embodiment of the present disclosure, the indicators of the method disclosed herein, are a number of immobilized conjugates comprising the label, such as forming a visually detectable line.

In another embodiment of the present disclosure, a major fraction of the sample is allowed to migrate from the sample loading zone to the test lines and contact the capturing agents without the presence of conjugates, such as before the conjugates.

In yet another embodiment of the present disclosure, said conjugates are allowed to migrate from the loading zone to the test lines and, if the target compound is bound to the capturing agent and the test line, are present, said conjugates bind to their respective test lines, thereby forming a detectable signal. In one embodiment of the present disclosure, said detectable signal indicates the presence of the target compound in the test sample.

Infectious disease

In one embodiment of the present disclosure, the target compound of the method disclosed herein, is a biomarker for an infectious disease. In another embodiment of the present disclosure, said infectious disease is a viral disease. In yet another embodiment of the present disclosure, said viral disease is a coronavirus infection. The coronavirus infection may be of the type SARS-CoV-2, also referred to as COVID-19.

In one embodiment of the present disclosure, the membrane layer of the method disclosed herein, comprises a test line that comprises or consists of an immobilized capturing agent specific for IgM antibodies, such as human IgM antibodies, such as a first test line. In another embodiment of the present disclosure, said membrane layer comprises a test line that comprises or consists of an immobilized capturing agent specific for IgG antibodies, such as human IgG antibodies, such as a second test line.

In yet another embodiment of the present disclosure, said membrane layer comprises a test line that comprises or consists of an immobilized capturing agent specific for a biomarker of a disease, such as disease-specific antibodies, such as a third test line.

IgG antibodies is the most common type of antibody found in the blood circulation. A person skilled in the art will know that IgM antibodies are the first antibodies to be produced in the response to an initial exposure to an antigen. Therefore, IgM levels in the blood are indicative of an active infection. IgG antibodies, on the other hand, are generated following class switching and maturation of the antibody response, thus they participate predominantly in the secondary immune response, thereby indicating a long-term response to an infection.

Sample

In one embodiment of the present disclosure, the sample comprises or consists of whole blood, serum, plasma, nasal secretions, sputum, urine, saliva and/or stool.

In one embodiment of the present disclosure, the sample comprises or consists of saliva.

In another embodiment of the present disclosure, the sample has been pre-treated, such as by mixing with a buffer solution, that may comprise anticoagulants. Anticoagulants are additives that inhibit for example blood and/or plasma from clotting, ensuring that the constituent that is to be measured, is non-significantly changed prior to the analytical process.

One important factor for the limited sensitivity, as compared to the other types of analytical methods, is that the format of the lateral flow devices poses a significant limitation on the sample. While lateral flow devices typically are limited to analysis of urine, blood, or water samples, other kinds of samples may in many cases be more readily accessible or may comprise a higher amount of the target analyte. However, the incompatibility between conventional lateral flow devices and specific sample types results in that these samples cannot be measured using conventional lateral flow devices, or at best, that the sensitivity is limited. Method simple

In a further aspect, the present invention relates to a method for detecting the presence of at least one target compound in a sample, the method comprising:

• providing a buffer solution;

• providing a lateral flow device comprising i. a membrane layer, comprising at least one test line, the at least one test line including an immobilized capturing agent for capturing the at least one target compound in the sample; ii. a sample loading zone for receiving the sample, the sample loading zone in fluidic connection with the test line;

• mixing the sample with the buffer solution, wherein the volumetric ratio between the buffer solution and the sample is between 5 parts buffer solution and 1 part sample and 15 part buffer solution and 1 part sample (between 5:1 and 15:1), thereby obtaining a sample solution;

• applying the sample solution to the sample loading zone;

• measuring the presence of the at least one target compound in the sample.

In a preferred embodiment of the present disclosure, the sample is a saliva sample. Obtaining saliva samples is associated with limited inconvenience for the patient, as compared to collection of other sample types, such as a nasopharyngeal sample. For example 0.1-1 ml of saliva may be rapidly collected from the mouth of the patient and has been shown to allow for sensitive quantitative and/or qualitative measurements of a target analyte in a sample. Typically an obtained saliva sample is between 0.1 and 0.2 ml, consequently it is a preference that the volume of the buffer solution is between 0.5 ml and 3 ml. Once the sample is mixed with the buffer solution, the obtained sample solution may be poured or dripped, e.g. pipetted, onto the sample loading zone.

The sample, preferably a saliva sample, is mixed with a buffer solution. The buffer solution may be selected to decrease non-specific binding when performing the test, dissolve components, such as mucous, of the sample, stabilizing the sample, and/or to adjust the viscosity of the sample.

The method of the present disclosure, as well as the disclosed kits provide several advantages over previously known methods. One advantage is that sensitive and highly specific measurements are possible without a need for a running buffer. A running buffer may be used, but the method and kits of the present disclosure can be used successfully without the need of adding a running buffer. Another advantage of the method and kits of the present disclosure is that aggregation in the sample is avoided by pre-treating the sample with a suitable buffer solution to obtain a sample solution. A further advantage of the method and kits of the present disclosure is that non-specific binding (NSB) to the immobilized capturing agent is minimized or even completely avoided. Furthermore, the method and kits of the present disclosure allow a more efficient detection of the presence of a target compound in a sample by minimizing, such as reducing, the waiting time required for the sample solution to migrate to the membrane layer comprising the immobilized capturing agent.

In another embodiment of the present disclosure, said method does not need the use of a running buffer. The presently disclosed method is capable of achieving highly sensitive and specific measurement without the need for a running buffer. Preferably the buffer solution is an aqueous solution and/or wherein the sample is mixed with the buffer solution at a volumetric ratio of 1 part sample to between 2 and 15 parts buffer solution, more preferably between 5 and 10 parts buffer solution, yet more preferably between 9 and 10 parts buffer solution. The buffer solution is advantageously mixed with the sample , as a buffer solution is mixed with the sample to obtain a sample solution, and the buffer solution present in the sample solution is sufficient to mobilize the analytes of the sample and/or the conjugates present at the conjugate zone, such as in the dry unused state.

In another embodiment of the present disclosure, the sample has been pre-treated, such as by mixing with a buffer solution that comprises sodium chloride, bovine serum albumin, and a borate buffer.

In another embodiment of the present disclosure, the sample has been pre-treated, such as by mixing with a buffer solution, that consists of sodium chloride, bovine serum albumin, and a borate buffer.

In another embodiment of the present disclosure, the sample has been pre-treated, such as by mixing with a buffer solution that comprises 1 M sodium chloride, 1% w/v bovine serum albumin, and 0.1 M borate buffer. It has been found that a buffer comprising at least one, preferably all, these components effectively reduces viscosity of the sample, in particular wherein the sample is saliva, and thereby helps in preventing formation of aggregates within the sample, such as within the sample solution.

In another embodiment of the present disclosure, the sample has been pre-treated, such as by mixing with a buffer solution that consists of 1 M sodium chloride, 1% w/v bovine serum albumin, and 0.1 M borate buffer.

In one embodiment of the present disclosure, the sample is diluted in the buffer solution by mixing between 5 parts buffer solution and 1 part sample and 15 part buffer solution and 1 part sample, thereby obtaining a sample solution.

In one embodiment of the present disclosure, the sample is diluted in the buffer solution by mixing 1 part sample and at least 5 parts buffer solution, more preferably at least 6 parts buffer solution, more preferably at least 7 parts buffer solution, more preferably at least 8 parts buffer solution, even more preferably at least 9 parts buffer solution, most preferably at least 10 parts buffer solution, such as 11 parts buffer solution, or 12 parts buffer solution, or even 13 parts buffer solution, or 14 parts buffer solution, such as 15 parts buffer solution. Preferably, the sample is diluted in the buffer solution by mixing 1 part sample and 10 parts buffer solution.

In one embodiment of the present disclosure, the sample is saliva, and it is diluted in the buffer solution by mixing between 5 parts buffer solution and 1 part sample and 15 part buffer solution and 1 part sample, preferably about 10 parts buffer solution and 1 part sample, thereby obtaining a sample solution.

In one embodiment of the present disclosure, diluting the sample in buffer solution as described herein is important for reducing the viscosity of the sample, for example wherein the sample is saliva, and thus preventing formation of aggregates within the sample, such as within the sample solution.

An important advantage of the method disclosed herein, wherein formation of aggregates within the sample is minimized or avoided, is that there is no or very low non-specific binding to the immobilized capturing agent in the membrane layer. Thus, the detection has high sensitivity and specificity. In one embodiment of the present disclosure, the membrane layer has not been provided with a blocking agent, such as after plotting the test and/or control line. This helps to minimize the time required for measurement, such to minimize and/or reduce the time sufficient for the sample solution to arrive at the test lines, and for the control line to be detectable, such as visually detectable. A common blocking agent is a solution comprising bovine serum albumin (BSA), which is typically applied to the membrane layer and allowed to dry. Other types of blocking agent are known to the person skilled in the art.

In one embodiment of the present disclosure, the capturing agents are configured for capturing a coronavirus, such as a human coronavirus, or fragments thereof.

In one embodiment of the present disclosure, the capturing agents are configured for binding to a spike protein, such as SARS-CoV S protein and/or SARS-CoV-2 S protein.

Thus, in one embodiment of the present disclosure, the method and/or the kits disclosed herein are suitable for diagnosis of an infectious disease, such as for diagnosis of COVID-19.

In yet an aspect, the present invention relates to a kit for detecting the presence of a target compound in a sample, the kit comprising:

• a buffer solution for diluting the sample, wherein said buffer solution comprises sodium chloride, bovine serum albumin, and a borate buffer; and

• a lateral flow device comprising:

• a membrane layer, comprising at least one test line, the at least one test line comprising an immobilized capturing agent for capturing the at least one target compound in the sample; and

• a sample loading zone for receiving the sample, the sample loading zone in fluidic connection with the test line.

The kit may advantageously be used for detecting the presence of a target compound in a saliva sample. In a preferred embodiment of the present disclosure, the kit is suitable for running the method as disclosed elsewhere herein.

Detailed description of drawings

The invention will in the following be described in greater detail with reference to the accompanying drawings. The drawings are exemplary and are intended to illustrate some of the features of the presently disclosed lateral flow devices, kits, and methods for detecting the presence of at least one target compound in a sample, and are not to be construed as limiting to the presently disclosed invention.

Fig. 1 shows schematic representations of a configuration of a lateral flow device according to a specific embodiment of the present disclosure. Fig. 1A shows a schematic representation of a lateral flow device (1) of the present disclosure. The device comprises a reagent zone (2), a membrane layer (3) and an absorption pad (4). The reagent zone comprising a sample loading zone (5) and a conjugate zone (6). For this specific lateral flow device, the conjugate zone does not comprise conjugates. Fig. 1 B shows the device during use, wherein a buffer solution (8) is applied to the lateral flow device, exemplified here by dipping said device into a receptacle (7) retaining the buffer solution comprising the conjugates. The buffer solution is thereby allowed to travel from the proximate end of the lateral flow device, to the distal end of the device.

Fig. 2 shows schematic representations of a configuration of a lateral flow device according to a specific embodiments of the present disclosure. Fig. 2A shows a schematic representation of a lateral flow device (1) of the present disclosure. The device comprises a reagent zone (2), a membrane layer (3) and an absorption pad (4). The reagent zone comprising a sample loading zone (5) and a conjugate zone (6). For this specific lateral flow device, the conjugate zone comprises conjugates. Fig 2B shows the lateral flow device during use, wherein a buffer solution (8) is applied to the lateral flow device, exemplified here by dipping said device into a receptacle (7) retaining the buffer solution. The buffer solution initially does not comprise conjugates, however upon contact with the conjugate zone, the conjugates are displaced by the buffer solution towards the absorption pad.

Fig. 3A shows schematic representation of a lateral flow device (1) comprising three test lines. The lateral flow device comprising a reagent zone (2) and a membrane layer (3). The test lines are here shown as visually observable and comprises a first test line (11), a second test line (12), and a third test line (13). The device furthermore comprises a control line (14). In a specific embodiment of the present disclosure the lateral flow device is targeting triaging of viral infections. For the three test line the first test line is configured to detect the presence of virus in the sample, thereby comprising a capturing agent specific for an epitope of the surface of the virus particle (19), the second test line is configured to detect the presence of IgM, the capturing agent may consequently be an anti-human IgM antibody (20), the third test line is configured to detect the presence of IgG, the capturing agent may consequently be anti-human IgG antibody (21). The control line is configured to target a conjugate, such as a non related control line conjugate, and may consequently be an antibody targeting this conjugate (22). Upon use, Fig. 3B, conjugates (15) may have been preloaded onto the lateral flow device. The conjugates comprising one or more conjugate protein binders (16) and a label (17), such as gold sol. Upon application of a blood sample (18), the sample flows mainly towards the test line and the distal end of the lateral flow device, thereby avoiding mixing with the conjugates. After a predetermined time, such as when the sample has reached the control line, the running buffer is provided to the conjugate zone, thereby acting to displace the conjugates. The conjugates may directly bind to an immobilized capturing agent, or bind to a target compound that has bound to an immobilized capturing agent, additionally test lines may be configured such that the conjugates does not bind, if a target compound has already bound to the test line.

Fig. 4 shows various outcomes of a viral infection test comprising three test lines, comprising a first test line (11) comprising anti-virus antibodies (19), such as anti COVID-19 antibodies, a second test line (12) comprising anti-human IgM antibodies (20), a third test line (13) comprising anti-human IgG antibodies (21) directed at capturing IgM, and a control line (14) comprising antibodies directed at capturing non- related control line conjugates (22). Following application of a sample and reconstitution of the conjugates by the running buffer, or the application of a running buffer comprising the conjugates, while a visually present control line indicates that the test has operated correctly, the presence of a visually present test line may either indicate the presence of the target compound or the absence of the target compound in the sample, depending on the configuration of the specific test line. For the first test line, binding of a virus particle, makes conjugates comprising synthetic fragments of virus as the conjugate protein binder not able to bind to the first test line, thereby producing an absence of a visual line. For the second and third test line, the conjugates may comprise conjugate protein binders directed at the respective target compounds, thereby producing visual lines if the target compound is present. While the test lines for human IgG and IgM antibodies function as a sandwich assay, where a visible line indicates the presence of these antibodies in the blood, the test line for the target compound, such as COVID-19, functions as a competitive assay, where a visible line indicates the absence of antibodies against the virus. In Fig. 4A the test line for the virus (11) is not visible, indicating the virus is present. Furthermore, the control line (14) is also visible, as well as the lines for IgM (12) and IgG (13) antibodies. In Fig. 4B, the control line and test line for human IgM antibodies are visible, suggesting an active infection, and that the test subject is infectious. In Fig. 4C, the control line, test lines for the target compound, and human IgG antibodies are present, suggesting that the test subject has had an infection (and no longer has antibodies against the target compound), and is now immune and non-infectious. In Fig. 4D the control line, test lines for the target compound, and test lines for human IgG and IgM antibodies are present on the lateral flow device, suggesting a previous infection, and that the test subject could be contagious. In Fig. 4E the control line and test line for the target compound are visible, suggesting that there is no active infection and that the test subject should be immunized. In Fig. 4F only the control line is visible, suggesting an active infection, but no immune reaction, indicating that the test subject is in urgent need of hospital care.

Examples

Example 1: Three line assay

Materials and Methods

A three line lateral flow device was fabricated comprising three test lines and one control line. The device comprised a membrane layer, wherein said three test lines and one control line were located. Said test lines and control lines comprising immobilized capturing agents for capturing COVID-19 virus particles, IgM, and IgG, Fig. 3A-B. The reagent zone was located at a proximate end of the device, and was in fluidic connection with the membrane layer. The sample was added to the sample loading zone, located between the conjugate zone and the test zone. Four whole blood samples were used, said samples comprising:

1. IgG and IgM

2. Negative control whole blood

3. Negative, dry format 4. IgG

Results

For sample 1 all four lines were visually present, for sample 2 only the virus test line and the control line were present, Fig 5A. Sample 3 shows only the control line while sample 4 shows a positive IgG.

Conclusions

The three test lines are able to bind their respective target compound. Example 2: Sensitive serum assay

Materials and methods

A similar device as in example 1. The test zone comprises immobilized S-protein, spike protein, (23) while the conjugates comprised anti-lgG gold sol (24) and anti-lgM gold sol (25), Fig. 6. Sample was added to the reagent zone prior to addition of conjugates. A negative control and five samples from NIBSC, WHO reference lab, of recovering patients screened using NIBSC ELISA assay were analysed.

Results

Conclusions

All samples were correctly identified, also sample 124 and 126 that were flagged as difficult for serological assays. Example 3: Virus test format

The coronavirus has multiple copies of the same epitope making it challenging to produce a virus assay in a conventional format. Mixing of the conjugate and the sample too early (before reaching the test zone) typically reduces the sensitivity and leads to aggregations of the gold label and may prevent further flow in the device.

A device comprising immobilized Anti-COVID-19 antibodies at the test line was used, Fig. 7. After the sample had been allowed to contact the test line, the conjugate solution was added comprising anti-covid-19 gold sol. The sample comprised trimeric S-Protein to mimic the Virus. Sample 1 was a negative serum sample, sample 2 was virus positive serum.

Results

Sample 1 resulted in no visual present test line, Fig. 8A. Sample 2 resulted in a visually present test line, Fig 8B.

Conclusions

The present sandwich assay format allows for the sample to interact with the test line prior to the conjugate particle interacting with said test line. Thereby, the conjugate particle is capable of binding to any present virus homologue.

Example 4 Fabrication of a lateral flow device Material and methods:

A lateral flow test device was fabricated by assembling components according to Fig.

9. Said figure illustrates a lateral flow test device (29) comprised of a nitrocellulose membrane (30) provided on an inert backing card (31). The membrane comprises a test line (32) and may advantageously further comprise a control line (33). The loading zone comprises two sample/conjugate pads (34), wherein at least one comprises conjugates, such as gold conjugates, for enabling a visual line at the test line or control line. Although not essential, the exemplified device comprises a Saliva filter pad (36) for filtering of the saliva sample and/or a sample solution comprising a saliva sample. Typically the saliva filter is a porous structure with pores in the size range between 0.1 and 1 pm. The device further comprises an end pad (37) for preventing leakage. The end pad may be provided, at the proximal end of the device, in a hydrophobic material. At the distal end of the device, across the test lines, is provided an absorbent pad (38).

The sample/conjugate pads were pre-treated with a buffer solution. Said solution was a tris buffered saline solution comprising BSA, PVP and Tween 20. The saliva filter pad was pre-treated with a PBS solution comprising BSA, NaCI and Tween 20. The nitrocellulose membrane was pre-treated (optional step) with a tris buffered saline solution comprising BSA, NaCI, BSA, sucrose and PVP.

Gold conjugates were fabricated by the following protocol:

• Obtain gold nanoparticles particles in Di water

• Dissolve anti-2019-nCoV-2 antibodies in the solution

• After all antibody has been added stir for 90 minutes

• Add 2% BSA in Borate buffer pH 7.5

• Stirred 60 minutes

• Centrifuge 6000 rpm for 5 minutes

• Dispose of supernatant resuspend in relevant OD in 1% BSA and 0.5% Tween

Plotting and blocking of the nitrocellulose membrane was performed by a standard plotter by plotting a solution of anti-SARS-CoV2 nucleocapsid protein mAb mixed with anti-2019-nCoV-2 antibody to give a total antibody concentration of 0.5 mg/ml. The nitrocellulose membrane was blocked by a TBS solution with NaCI, BSA, Sucrose and PVP.

Conclusion

A lateral flow device for diagnosing COVID-19 by detection of protein antigen from SARS-CoV-2 in saliva samples was fabricated.

Example 5 Detection of protein antigen from SARS-CoV-2 in saliva samples from individuals suspected of COVID-19

The lateral flow test used was an immuno-chromatographic membrane assay that uses highly sensitive antibodies to detect SARS-CoV-2 Spike protein from saliva specimens. A SARS-CoV-2 specific antibody and a control antibody are immobilized onto a membrane support as two distinct lines and combined with other reagents/pads to construct a test strip, as shown in Fig. 10. The figure illustrates a lateral flow test device (29) comprising a nitrocellulose membrane (30), on an inert backing card (31). The membrane comprises a test line (32) and a control line (33). The device has a sample/conjugate pad (34) comprising gold conjugates (35), an (optional) saliva filter pad (36), and a sink pad/absorbent pad (38).

The lateral flow test strip consists of: 1. A backing card structure to support the different layers

2. A sample/conjugate pad, where the mobile phase of the assay is sprayed.

3. A nitrocellulose membrane comprising the solid phase of the assay, which comprises two distinct areas, one where the test line is plotted and one where the control line is plotted.

4. A sink pad

Both the sample/conjugate and saliva filter pads are treated in order to compensate the pH effect form the saliva sample and remove or eliminate any non-specific interactions. The mobile phase consisted of two parts:

1. An anti-2019 nCoV (S1) 40nm gold conjugate and

2. A goat anti-mouse IgG conjugate

Both conjugates are diluted in a gold spraying buffer, containing high amounts of sucrose and protein, in order to enrich the particles’ surface and improve the stability of the conjugates. Both gold conjugates are sprayed at a concentration of 10 OD each.

The nitrocellulose is plotted with anti-2019 nCoV (S1) to form the test line and rabbit anti mouse IgG to form the control line. After the plotting the NC is being blocked in order to remove any non-specific binding and aggregation of the gold conjugate.

A saliva sample is added to the lateral flow device and the test line is visually inspected after 30 minutes.

Subjects that are both positive and negative for COVID-19 infection, as measured with a gold standard method, are tested using the lateral flow test strip. For the subjects that are determined to have an ongoing COVID-19 infection, two visual lines appear shortly after adding the saliva sample to the test line, the first visual line indicating that the virus particles are correctly detected and the second visual line (control line) indicating the validity of the test. For the subjects that are determined to not have an ongoing COVID-19 infection, a single visual line appears shortly after adding the saliva sample to the test line, the single visual line (control line) indicating the validity of the test.

Conclusion

The lateral flow test is capable of correctly diagnosing COVID-19 by detection of protein antigen from SARS-CoV-2 in saliva samples.

Example 6 Evaluation of Covid-19 lateral flow test LOD and specificity using filtered spiked saliva (using salivate)

Preparing serial dilutions

The SARS-CoV-2 isolate REMRQ0001/Human/2020/Liverpool is used for the serial dilutions and Vero E6 cells (C1008; African green monkey kidney cells) are used to propagate the virus. Cells are maintained in Dulbecco’s minimal essential medium (DM EM) containing 10% foetal bovine serum (FBS) and 0.05 mg/ml gentamicin at 37°C with 5% C02 and FBS concentration was reduced to 4% for viral propagation.

Ten-fold serial dilutions of virus stock starting at 10⁶ pfu/ml are made using DMEM media as a diluent. Saliva is collected using salivates. Serial dilutions are made with saliva at a final concentration of 10⁶, 10⁵, 10⁴, 10³ and 10² pfu/ml. Three lots are tested and 10 replicates per lot are used. Saliva of the negative donors is used as negative controls.

Test evaluation

The measurements are carried out on COVID-19 Saliva lateral flow tests. For the positive samples, a test will be considered a fail if no control line is visible after 30 minutes.

Percentage sensitivity will be calculated by the following formula: Sensitivity = (Observed Positive Results/Actual Positive Results) x100

Intra-lot precision is a measurement of the variation within a single batch of COVID-19 Saliva Tests. It can be calculated using the following formula: Precision Intra- lot=(Observed Results/Expected Results)x100

In this validation protocol the mean results from each lot of assays at each concentration (ran during the validation study for limit of detection and dynamic range) will be compared and percentage standard error calculated for the three lots.

Evaluation of cross reactivity and interference

The specificity of the COVID-19 saliva test is evaluated by testing potentially cross reactive microorganisms. The microorganisms are tested in triplicate in the absence (cross-reactivity) or presence of SARS-CoV-2 virus (interference) in saliva samples.

The microorganisms include Adenovirus, Human Metapneumovirus (hMPV), Parainfluenza virus 1-4, Influenza A & B, Enterovirus, Respiratory syncytial virus, Rhinovirus, Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus pyogenes, Candida albicans, Pooled human nasal wash, Bordetella pertussis, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Staphylococcus aureus, Staphylococcus epidermidis

Example 7 Evaluation of Covid-19 virus epitope sensitivity

Lateral flow tests were fabricated comprising a porous phase of nitrocellulose, a sample pad and an absorbent pad. The test line was either sprayed with Anti-2019-n-CoV(S1) targeting the more prominent S protein on the surface of the virus, and Hu mAb to SAR- CoV-2 nucleocapsid protein targeting a less prominent N protein inside the virus.

The test lines were sprayed by a solution comprising either of the two antibodies at various concentrations between 0.1 and 5 mg/ml.

Saliva samples positive for Covid-19 were tested, and it was concluded that devices having a test line directed at the S protein of the virus had better sensitivity than those that were directed at the N protein.

Example 8 Optimization of Lateral flow tests

Lateral flow tests were fabricated comprising a porous phase of nitrocellulose, a sample pad and an absorbent pad. The test line was sprayed with Anti-2019-n-CoV(S1) targeting the more prominent S protein on the surface of the virus. The control line was Ms mAb to human IgG.

Optimization of test line and control line concentration

Measurements were performed at multiple concentrations for the test and control line and the most sensitive results were seen at 2.4 mg/ml test line and 2.0 mg/ml control line. These concentrations were acquired by diluting the COVID antibody down to 1.2 mg/ml in PBS 0.1% BSA buffer, then both the test and control line were sprayed down twice. These concentrations showed the best signals and no NSB when run with distilled water.

Optimization of saliva buffer

The saliva mix buffer formula was further optimized by testing buffers with different amounts of protein, salt and detergents. The optimal results were obtained by 0.1 M borate buffer with 1M NaCI and 1% BSA. Multiple different formulas were assessed and this one resulting in no measurable non-specific binding. The ratio of the saliva mix buffer was also tested. Despite the high dilution of the saliva, a dissolution ratio between the saliva and buffer of 1:10 worked the best (one part saliva). The results are provided in the table below.

Optimization of run speed

The time until development of test lines and control lines were measured with lateral flow tests, manufactured according to above, where blocking of the nitrocellulose was performed following plotting of the lines, and were no blocking of the nitrocellulose was performed following plotting of the lines. It was concluded that the lateral flow devices that did not have blocking developed control and test lines significantly faster than the others. At the same time the sensitivity and specificity remained at the same level as with blocking. Analysis of lateral flow test sensitivity

Five different saliva samples were spiked with known concentrations of virus and tested in triplicates. All the samples before being diluted were showing non-specific binding. When diluted 1 in 10 in the saliva mix buffer there was no non-specific binding present. The results are provided in the table below.

Items

1. A method for detecting the presence of at least one target compound in a sample, the method comprising: a. providing a buffer solution; b. providing a lateral flow device comprising i. a membrane layer, comprising at least one test line, the at least one test line including an immobilized capturing agent for capturing the at least one target compound in the sample; ii.a sample loading zone for receiving the sample, the sample loading zone in fluidic connection with the test line; c. mixing the sample with the buffer solution, thereby obtaining a sample solution; d. applying the sample solution to the sample loading zone; e. measuring the presence of the at least one target compound in the sample.

2. The method according to item 1, wherein the sample comprises or consists of whole blood, serum, plasma, nasal secretions, sputum, urine, saliva and/or stool, preferably wherein the sample is a saliva sample.

3. The method according to any one of the preceding items, wherein the volumetric ratio between the buffer solution and the sample is between 5 parts buffer solution and 1 part sample and 15 part buffer solution and 1 part sample.

4. The method according to any one of the preceding items, wherein the volumetric ratio between the buffer solution and the sample is 1 part sample and at least 5 parts buffer solution, more preferably at least 6 parts buffer solution, more preferably at least 7 parts buffer solution, more preferably at least 8 parts buffer solution, even more preferably at least 9 parts buffer solution, most preferably at least 10 parts buffer solution, such as 11 parts buffer solution, or 12 parts buffer solution, or even 13 parts buffer solution, or 14 parts buffer solution, such as 15 parts buffer solution.

5. The method according to any one of the preceding items, wherein the buffer solution comprises sodium chloride, bovine serum albumin, and a borate buffer.

6. The method according to item 5, wherein the buffer solution comprises 1 M sodium chloride, 1% w/v bovine serum albumin, and 0.1M borate buffer.

7. The method according to any one of the preceding items, wherein the membrane layer has not been provided with a blocking agent, such as after plotting the test and/or control line.

8. The method according to any one of the preceding items, wherein the capturing agents are configured for capturing a coronavirus, such as a human coronavirus, or fragments thereof.

9. The method according to item 8, wherein the capturing agents are configured for binding to a spike protein, such as SARS-CoV S protein and/or SARS-CoV- 2 S protein.

10. The method according to any one of the preceding items, wherein the lateral flow device comprises a conjugate zone, and wherein said conjugate zone comprises in a dry unused state conjugates for binding to the target compound and/or the immobilized capturing agent.

11. The lateral flow device according to any one of the preceding items, wherein the membrane layer comprises a control line.

12. The lateral flow device according to any one of the preceding items, wherein the control line comprises a further immobilized capturing agent specific for binding to the conjugate.

13. The method according to any one of the preceding items, further comprising waiting a sufficient amount of time for the sample solution to arrive at the test lines. 14. The method according to any one of the preceding items, wherein the sufficient amount of time is the time lapsed until the control line is expected to be detectable, such as visually detectable.

15. The method according to any one of the preceding items, wherein measuring the presence of the at least one target compound in the sample comprises detecting for each test line, the presence of at least one indicator, such as a visible line.

16. The method according to any one of the preceding items, wherein the indicators are a number of immobilized conjugates comprising the label, such as forming a visually detectable line.

17. The method according to any one of the preceding items, wherein a major fraction of the sample is allowed to migrate from the sample loading zone to the test lines and contact the capturing agents without the presence of conjugates.

18. The method according to any one of the preceding items, wherein the detectable signal indicates the presence of the target compound in the test sample.

19. The method according to any one of the preceding items, wherein the membrane layer comprises a test line that comprises or consists of an immobilized capturing agent specific for IgM antibodies, such as human IgM antibodies, such as a first test line.

20. The method according to any one of the preceding items, wherein the membrane layer comprises a test line that comprises or consists of an immobilized capturing agent specific for IgG antibodies, such as human IgG antibodies, such as a second test line.

21. The method according to any one of the preceding items, wherein the membrane layer comprises a test line that comprises or consists of an immobilized capturing agent specific for a biomarker of a disease, such as disease-specific antibodies, such as a third test line. 22. The method according to any one of the preceding items, wherein the lateral flow device is configured for operation as a sandwich assay and/or as a competitive assay.

23. A kit for detecting the presence of a target compound in a sample, the kit comprising: a. a buffer solution for diluting the sample, wherein said buffer solution comprises sodium chloride, bovine serum albumin, and a borate buffer; and b. a lateral flow device comprising: i. a membrane layer, comprising at least one test line, the at least one test line comprising an immobilized capturing agent for capturing the at least one target compound in the sample; ii. a sample loading zone for receiving the sample, the sample loading zone in fluidic connection with the test line.

24. The kit according to item 23, wherein said kit is suitable for running the method according to any one of items 1 to 22.

Claims

1. A lateral flow device for detecting the presence of at least one target compound in a saliva sample, the lateral flow device comprising:

• a membrane layer, comprising at least one test line, the at least one test line including an immobilized capturing agent for capturing the at least one target compound in the sample;

• a reagent zone, comprising: i. a conjugate zone for receiving a running buffer, and ii. a sample loading zone for receiving the sample; wherein the sample loading zone is located, along the axial length of the lateral flow device, between the conjugate zone and the at least one test line, and wherein the target compound is a human coronavirus or a fragment thereof.

2. The lateral flow device according to claim 1, wherein the conjugate zone, in the dry unused state, comprises at least one conjugate.

3. The lateral flow device according to claim 1, wherein the conjugate zone does not comprise conjugates.

4. The lateral flow device according to any one of the preceding claims, wherein the sample loading zone is at least partially covered by a sample filter, for filtering the saliva sample.

5. The lateral flow device according to claim 4, wherein the sample filter comprises pores with a pore size between 0.1 and 5 pm, preferably between 0.1 pm and 1 pm.

6. The lateral flow device according to any one of the preceding claims, wherein the conjugate comprises a label and at least one conjugate protein binder, such as multiple conjugate protein binders.

7. The lateral flow device according to claim 6, wherein the label is a metal nanoparticle, such as a gold nanoparticle, and/or a coloured latex particle.

8. The lateral flow device according to any one of claims 6-7, wherein the conjugate protein binder is selected from the list including an antibody, an Ig unit, an aptamer, an affimer, an RNA molecule, a DNA molecule, an organic polymer, or a fragment thereof.

9. The lateral flow device according to claim 8, wherein the organic polymer is a molecularly imprinted polymer.

10. The lateral flow device according to any one of the preceding claims, wherein the membrane layer comprises multiple test lines, for capturing multiple target compounds in the sample.

11. The lateral flow device according to any one of the preceding claims, wherein each test line comprises a different immobilized capturing agent.

12. The lateral flow device according to any one of the preceding claims, wherein each test line is configured such that it can detect the presence of a different target compound.

13. The lateral flow device according to any one of the preceding claims, wherein the membrane layer comprises three test lines, said test lines having immobilized capturing agent specific for capturing human IgM antibodies, human IgG antibodies, and human coronavirus particles, or fragments thereof, respectively.

14. The lateral flow device according to any one of the preceding claims, wherein the lateral flow device is configured for operation as a sandwich assay and/or as a competitive assay.

15. The lateral flow device according to any one of the preceding claims, wherein the membrane layer directly contacts the test line.

16. The lateral flow device according to any one of the preceding claims, wherein the conjugate zone is located at a proximate end of the lateral flow device.

17. The lateral flow device according to any one of the preceding claims, wherein said lateral flow device further comprises an absorbent pad, located at a distal end of the lateral flow device.

18. The lateral flow device according to any one of the preceding claims, wherein the reagent zone comprises at least one application pad, the application pad comprising the conjugate zone and/or the sample loading zone, such as a conjugate pad and/or a sample pad.

19. The lateral flow device according to any one of the preceding claims, wherein said membrane layer comprises the reagent zone.

20. The lateral flow device according to any one of the preceding claims, wherein said lateral flow device further comprises a backing layer in an inert material.

21. The lateral flow device according to any one of the preceding claims, wherein the membrane layer comprises or consists of nitrocellulose, cellulose acetate or paper.

22. The lateral flow device according to any one of the preceding claims, wherein the lateral flow device comprises a cassette, and/or wherein the lateral flow device is provided as a dipstick.

23. The lateral flow device according to any one of the preceding claims, wherein the capturing agent comprises a capture protein binder.

24. The lateral flow device according to claim 23, wherein the capture protein binder is selected from the list including an antibody, an aptamer, an affimer, an RNA molecule, a DNA molecule, an organic polymer, the target compound, a derivative of the target compound, or a fragment thereof.

25. The lateral flow device according to claim 24, wherein the organic polymer is a molecularly imprinted polymer.

26. The lateral flow device according to any one of the preceding claims, wherein the capturing agent and/or conjugate is specific for an epitope of the target compound.

27. The lateral flow device according to claim 26, wherein the target compound comprises multiple copies of the epitope.

28. The lateral flow device according to any one of the preceding claims, wherein the membrane layer comprises a control line.

29. The lateral flow device according to claim 28, wherein the control line comprises a further immobilized capturing agent specific for binding to the conjugate.

30. A kit for detecting the presence of a target compound in a sample, the kit comprising:

• a running buffer; and

• a lateral flow device comprising: i. a membrane layer, comprising at least one test line comprising an immobilized capturing agent specific for capturing the at least one target compound in the sample, ii. a reagent zone comprising:

• a conjugate zone for receiving the running buffer, and

• a sample loading zone for receiving the sample; wherein the sample loading zone is located, along the axial length of the lateral flow device, between the conjugate zone and the at least one test line, and wherein the target compound is a human coronavirus or a fragment thereof.

31. The kit according to claim 30, wherein the lateral flow device is configured as in any one of claims 1-29.

32. The kit according to any one of claims 30-31 , wherein the conjugate zone, in the dry unused state, comprises conjugates.

33. The kit according to any one of claims 30-31 , wherein conjugates are provided as part of the running buffer, such as wherein the kit is configured to have conjugates added to the lateral flow device together with the running buffer.

34. The lateral flow device according to any one of claims 30-33, wherein the sample loading zone is at least partially covered by a sample filter, for filtering the saliva sample.

35. The lateral flow device according to claim 34, wherein the sample filter comprises pores with a pore size between 0.1 and 5 pm, preferably between 0.1 pm and 1 pm.

36. The kit according to any one of claims 30-35, wherein the kit is configured such that upon application of the running buffer to the lateral flow device, the conjugates will flow substantially with the running buffer, such as from a proximate end of the lateral flow device to a distal end of the lateral flow device.

37. The kit according to any one of claims 30-36, wherein the kit comprises a receptacle for retaining the running buffer.

38. The kit according to any one of claims 30-37, wherein the kit is configured such that the running buffer may be applied to an end of the lateral flow device, such as the proximate end, by pipetting, and/or such that the running buffer may be applied to the lateral flow device by dipping an end of said lateral flow device, such as the proximate end, in the receptacle.

39. The kit according to any one of claims 30-38, wherein the kit further comprises a buffer solution for pre-treating the sample, before application onto the sample loading zone.

40. The kit according to any one of claims 30-39, wherein the buffer solution comprises sodium chloride, bovine serum albumin, and a borate buffer.

41. The kit according to any one of claims 30-40, wherein the buffer solution comprises 1 M sodium chloride, 1% w/v bovine serum albumin, and 0.1 M borate buffer.

42. A method for detecting the presence of at least one target compound in a sample, the method comprising:

I. providing a kit comprising: • a running buffer; and

• a lateral flow device comprising:

- a membrane layer comprising at least one test line comprising an immobilized capturing agent for capturing the at least one target compound in the sample, wherein the at least one target compound is human coronavirus or a fragment thereof;

- a reagent zone comprising:

• a conjugate zone for receiving the running buffer; and

II. applying the sample to the sample loading zone;

III. applying the running buffer to the conjugate zone;

V. detecting, for each test line, the presence of at least one indicator, such as a visible line; thereby detecting the presence of at least one target compound in the sample.

43. The method according to claim 42, wherein the kit is configured according to any one of claims 30-41, and/or wherein the lateral flow device is configured according to any one of claims 1-29.

44. The method according to any one of claims 42-43, further comprising application of the running buffer to the sample loading zone after the step of adding the sample and before the step of adding running buffer to the conjugate zone.

45. The method according to any one of claims 42-44, wherein the kit, the application of the sample and/or the application of the running buffer is configured such that a substantial fraction of the sample is allowed to flow over the test line before the conjugates.

46. The method according to any one of claims 42-45, wherein the kit comprises conjugates for binding to the target compound and/or the immobilized capturing agent, said conjugates being, in the dry unused state, provided at conjugate zone or as part of the running buffer

47. The method according to any one of claims 42-46, wherein the lateral flow device is provided with the conjugates present at the conjugate zone, such as in a dry unused state.

48. The method according to any one of claims 42-47, wherein the running buffer does not comprise conjugates.

49. The method according to any one of claims 42-48, wherein the lateral flow device is provided without the conjugates present at the conjugate zone.

50. The method according to claim 49, wherein the running buffer comprises the conjugates.

51. The method according to any one of claims 42-50, wherein the running buffer is applied to the loading zone, and/or a proximate end of the lateral flow device, at or near the conjugate zone.

52. The method according to any one of claims 42-51 , wherein the running buffer is first applied to an area between the sample loading zone and the conjugate zone, followed by a second application of running buffer to the conjugate zone and/or the proximate end of the lateral flow device, wherein the first application is carried out for a predetermined time and/or consists of application of a predetermined volume.

53. The method according to any one of claims 42-52, wherein the running buffer is applied by dipping an end of the lateral flow device in the running buffer.

54. The method according to any one of claims 42-53, wherein the running buffer is applied only after the sample has been allowed to interact with the at least one test line.

55. The method according to any one of claims 42-54, wherein the sufficient time is the time lapsed until the control line is expected to be detectable, such as visually detectable.

56. The method according to any one of claims 42-55, wherein the indicators are a number of immobilized conjugates comprising the label, such as forming a visually detectable line.

57. The method according to any one of claims 42-56, wherein a major fraction of the sample is allowed to migrate from the sample loading zone to the test lines and contact the capturing agents without the presence of conjugates.

58. The method according to any one of claims 42-57, wherein the conjugates are allowed to migrate from the loading zone to the test lines and, if the target compound is bound to the capturing agent and the test line, are present, said conjugates bind to their respective test lines, thereby forming a detectable signal.

59. The method according to any one of claims 42-58, wherein the detectable signal indicates the presence of the target compound in the test sample.

60. The method according to any one of claims 42-59, wherein the target compound is a biomarker for an infectious disease.

61. The method according to claim 60, wherein the infectious disease is a viral disease.

62. The method according to claim 61, wherein the viral disease is a coronavirus infection.

63. The method according to any one of claims 42-62, wherein the membrane layer comprises a test line that comprises or consists of an immobilized capturing agent specific for IgM antibodies, such as human IgM antibodies, such as a first test line.

64. The method according to any one of claims 42-63, wherein the membrane layer comprises a test line that comprises or consists of an immobilized capturing agent specific for IgG antibodies, such as human IgG antibodies, such as a second test line.

65. The method according to any one of claims 42-64, wherein the membrane layer comprises a test line that comprises or consists of an immobilized capturing agent specific for a biomarker of a disease, such as disease-specific antibodies, such as a third test line.

66. The method according to any one of claims 42-65, wherein the sample has been pre-treated, before application to the sample loading zone, such as by mixing with a buffer solution comprising anticoagulants.

67. The method according to any one of claims 42-66, wherein the sample is mixed with a buffer solution before application to the sample loading zone.

68. The method according to any one of claims 42-67, wherein the volumetric ratio between the buffer solution and the sample is between 5 parts buffer solution and 1 part sample and 15 part buffer solution and 1 part sample, such as wherein the ratio of sample to buffer solution is between 1:5 and 1:15.

69. The method according to any one of claims 42-68, wherein the volumetric ratio between the buffer solution and the sample is between 7 parts buffer solution and 1 part sample and 12 part buffer solution and 1 part sample, such as wherein the ratio of sample to buffer solution is between 1:7 and 1:12.

70. The method according to any one of claims 42-69, wherein the buffer solution comprises sodium chloride, bovine serum albumin, and a borate buffer.

71. The method according to any one of claims 42-70, wherein the buffer solution comprises 1 M sodium chloride, 1% w/v bovine serum albumin, and 0.1 M borate buffer.