WO2022058526A1 - Procédés pour déterminer l'état d'infectivité du coronavirus - Google Patents

Procédés pour déterminer l'état d'infectivité du coronavirus Download PDF

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Publication number
WO2022058526A1
WO2022058526A1 PCT/EP2021/075660 EP2021075660W WO2022058526A1 WO 2022058526 A1 WO2022058526 A1 WO 2022058526A1 EP 2021075660 W EP2021075660 W EP 2021075660W WO 2022058526 A1 WO2022058526 A1 WO 2022058526A1
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Prior art keywords
coronavirus
protein
binding species
specific
viral
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PCT/EP2021/075660
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English (en)
Inventor
Peter Fitzgerald
Ivan McConnell
John Lamont
Jim Curry
Philip Lowry
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Randox Laboratories Ltd
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Priority to EP21782657.7A priority Critical patent/EP4214511A1/fr
Publication of WO2022058526A1 publication Critical patent/WO2022058526A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)

Definitions

  • Coronavirus disease 2019 (Covid-19) which is caused by SARS-CoV-2 viral infection was declared a pandemic by the World Health Organisation on 11th March 2020. Since the first cases were identified in humans in late 2019 there have been over 30 million confirmed cases and over 940,000 deaths worldwide as of 18th September 2020, many regions are still struggling to contain the ongoing pandemic.
  • SARS- CoV-2 is believed to be primarily spread via respiratory droplets (released during coughing, sneezing and talking) that are transmitted to persons in close contact with an infected individual.
  • Wblfel et al [1] reported that infectious virus could be detected in nasopharyngeal/oropharyngeal swabs collected during the first week of infection, but not in swabs taken after this period, despite high rates of SARS-CoV-2 RNA being detected at these later time points.
  • Asymptomatic individuals have also been found to be infectious.
  • the current invention provides novel methods for distinguishing infectious and non- infectious individuals and means to enhance currently available detection methods.
  • the current invention provides a method for identifying coronavirus virions in an in vitro sample, the method comprising the steps of (a) contacting said sample with two or more of the following capture binding species; an antibody specific to a viral surface protein, an aptamer specific to a viral surface protein and a virus receptor (b) contacting any virions bound to the capture binding species with a detection binding species selected from an antibody specific to a viral surface protein, an aptamer specific to a viral surface protein and a virus receptor and (c) detecting or determining the presence or amount of coronavirus virion.
  • a second aspect of the current invention provides a method which further comprises additional steps for identifying coronavirus internal viral proteins in the sample as well as intact coronavirus virions, said steps comprising (a)(i) contacting the sample with one or more of the following capture binding species; an antibody specific to an internal viral protein and an aptamer specific to an internal viral protein (b)(i) contacting any viral protein bound to the capture species with a detection binding species selected from an antibody specific to a viral surface protein, an antibody specific to an internal viral protein, an aptamer specific to a viral surface protein and an aptamer specific to an internal viral protein and (c) (i) detecting or determining the presence or amount of coronavirus internal viral protein.
  • a third aspect of the current invention is the use of any methods of the invention to aid in the determination of the coronavirus infectivity status of an individual by detecting the presence or amount of intact coronavirus virion and/or coronavirus internal viral protein.
  • a fourth aspect of the current invention is a method for enhancing current PCR detection methods by first isolating coronavirus virion from a sample.
  • Figure 1 A schematic showing the various options for a multiplex format to determine the infectiousness of an individual.
  • Numbers 1-3 show capture binding species attached to a substrate which are specific to the Spike protein receptor binding domain (RBD) of SARS-CoV-2.
  • RBD Spike protein receptor binding domain
  • (1) represents an anti-spike protein RBD IgG antibody
  • (2) represents an ACE2 receptor
  • (3) represents an antispike protein RBD aptamer.
  • These three types of capture binding species are indicative of infectious SARS-CoV-2 virion.
  • Numbers 4 and 5 show capture binding species attached to a substrate which are specific to the nucleocapsid protein of SARS-CoV-2.
  • (4) represents an anti-NP IgG antibody and (5) represents an anti-NP aptamer.
  • capture binding species can be indicative of non- infectious SARS-CoV-2 fragments.
  • detection is facilitated by the addition of anti-spike protein RBD and anti-NP Fabs.
  • the detection binding species can be an antibody, a receptor or an aptamer.
  • Such assays can be calibrated with recombinant spike protein RBD or recombinant NP.
  • Figure 2 A schematic showing the initial process for ascertaining the viral infectivity status of an individual by isolating the coronavirus virion by way of anti-spike binding species and the nucleocapsid protein (NP) by of anti-nucleocapsid binding species (in the schematic antibodies specific to each of the protein targets are used as illustrative binding species).
  • the key advantage of the current invention is the ability to isolate and identify virions in a sample which are indicative of an active infection, but also importantly to indicate individuals who are most likely to be infectious to others.
  • the methods described herein can be used in isolation as a rapid test or can be integrated into and enhance current viral RNA tests.
  • the current invention provides a method for identifying coronavirus virions in an in vitro sample, the method comprising the steps of (a) contacting said sample with two or more of the following capture binding species; an antibody specific to a viral surface protein, an aptamer specific to a viral surface protein and a virus receptor (b) contacting any virions bound to the capture binding species with a detection binding species selected from an antibody specific to a viral surface protein, an aptamer specific to a viral surface protein and a virus receptor and (c) detecting or determining the presence or amount of coronavirus virion.
  • the advantage of this method is that if the virion is detected in a sample an individual can be classified as infective and appropriate measures can quickly be taken to ensure that their infectiousness is minimised.
  • the amount of virion detected can indicate the degree to which the individual is infectious, and their classification can be adjusted based on this.
  • a further advantage of virion capture by binding ligands which target different antigenic epitopes of the viral surface protein, for example the virus spike protein, would theoretically be greater specificity and less false positives compared to standard antigen tests. For example, virion binding to the viral receptor will do so in the open spike protein conformation. This may be particularly useful for detection of SARS-CoV-2 strains possessing the D614G mutation which has been reported to enhance the propensity of the virion to exist in an open conformation. By comparing the ratios and/or absolute amounts of the virions captured by the different binding ligands, further information regarding the infectivity status of the individual can be gathered.
  • coronavirus refers to any member of the family Coronaviridae, preferably members of the subfamily Orthocoronavirinae belonging to the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus.
  • the methods of the invention may be applied to any Coronavirus currently known or indeed any future coronavirus identified since it relies on detecting proteins, and optionally genetic material, which are characteristic of the family Coronaviridae.
  • the Coronavirus is a Betacoronavirus selected from Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (Mers-CoV) or Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); even more preferably the Coronavirus is SARS-CoV-2.
  • SARS-CoV Severe acute respiratory syndrome coronavirus
  • Mers-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2
  • the Coronavirus is SARS-CoV-2.
  • virion refers to the complete, infective form of a virus found within a sample and comprising a core of RNA and a capsid.
  • coronavirus virion refers to the complete infective form of a coronavirus found within a sample.
  • the preferred virions of the invention are SARS-CoV-2 virions and the capture binding species are selected from an antibody specific to a SARS-CoV-2 surface protein, an aptamer specific to a SARS-CoV-2 surface protein and a SARS-CoV-2 receptor.
  • viral surface protein refers to any protein which is located on the surface of a coronavirus virion and which is accessible to a binding species when the virion is intact. This includes the spike protein, the envelope protein (E) and the membrane protein (M).
  • the preferred viral surface protein target of the invention is the spike protein, more preferably the receptor-binding domain of the spike SI protein domain of SARS-CoV-2.
  • virus receptor refers to any receptor which a virus can use to facilitate its entry into a host cell. It preferably refers to virus receptor proteins.
  • virus receptor proteins include angiotensin-converting enzyme 2 (ACE2), aminopeptidase N (APN), dipeptidyl peptidase 4 (DPP4) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1).
  • ACE2 angiotensin-converting enzyme 2
  • APN aminopeptidase N
  • DPP4 dipeptidyl peptidase 4
  • CEACAM1 carcinoembryonic antigen-related cell adhesion molecule 1
  • Binding proteins can be isolated native proteins, recombinant proteins or modifications thereof which retain their function. Binding domains of coronaviruses can also recognise glycans, so glycoproteins also fall under the definition of a virus receptor in the current invention.
  • a virus receptor can also be a synthetic receptor such as a molecular imprinted polymer.
  • capture binding species refers to any entity which has affinity to a coronavirus virion or internal viral protein, and which is used in the initial binding of a target in an in vitro sample.
  • Preferred capture binding species include antibodies to coronavirus surface proteins, aptamers to coronavirus surface proteins, coronavirus receptors, antibodies to coronavirus internal proteins or aptamers to coronavirus internal proteins.
  • Coronavirus proteins are known to be glycosylated and the capture binding species of the invention may also be a lectin which binds to a viral envelope glycan, particularly plant lectins which have affinity for mannose and N- acetylglucosamine sugar moieties.
  • detection or determination may be direct and make use of electrical conductance, surface plasmon resonance or Raman spectroscopy-based methods, or it may require the addition of a detection binding species.
  • detection binding species refers to any entity which has affinity to a coronavirus virion orfragment thereof and which is used in the detection or determination of its target bound to a capture binding species. Detection binding species may also be conjugated to various reporter moieties for detection and/or determination of a target, including but not limited to radionuclides, fluorophores, dyes or enzymes including, for example, horse-radish peroxidase and alkaline phosphatase. Preferred detection binding species include antibodies to coronavirus surface proteins, aptamers to coronavirus surface proteins, coronavirus receptors, antibodies to coronavirus internal proteins or aptamers to coronavirus internal proteins. "Detecting" or “detection” as referred to herein means qualitatively analysing for the presence or absence of a target, while “determining” or “determination” means quantitatively analysing for the amount of a target.
  • antibody refers to an immunoglobulin which specifically recognises an epitope on a target as determined by the binding characteristics of the immunoglobulin variable domains of the heavy and light chains (VHS and VLS), more specifically the complementaritydetermining regions (CDRs).
  • VHS and VLS immunoglobulin variable domains of the heavy and light chains
  • CDRs complementaritydetermining regions
  • Many potential antibody forms are known in the art, which may include, but are not limited to, monoclonal antibodies or polyclonal antibodies, antibody fragments (for example Fab, Fab', and Fv fragments, linear antibodies, single chain antibodies (e.g.
  • references to antibodies in the context of the present invention refer to polyclonal or monoclonal antibodies, antibody fragments or ScFvs.
  • Antibodies may also be conjugated to various reporter moieties for detection and/or determination of a target, including but not limited to radionuclides, fluorophores, dyes or enzymes including, for example, horse-radish peroxidase (HRP) and alkaline phosphatase.
  • HRP horse-radish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • the capture and/or detection binding species are Fab or ScFv antibodies specific to a viral surface protein.
  • An advantage of using such binding species is that they are smaller and therefore a greater number can bind to a single virion, hence increasing the assay sensitivity.
  • the detection can be direct or a detection binding species can be added.
  • a Fab or ScFv is used as the detection binding species following capture of a target protein they can increase sensitivity in comparison to using a larger detection binding species.
  • a single ScFv can be used as the detection binding species for several different capture binding species, for example a capture antibody, aptamer and viral receptor.
  • the capture and detection species for a target can be the same or be different, they can bind to the same epitope on the target or to different epitopes.
  • the capture binding species and detection binding species have the same coronavirus epitope binding specificities.
  • An advantage of this is that the same antibody can be used for capture and detector thus reducing development costs and simplifying the assay. This can be applied to the multiplex format. For example, when capture is with a spike protein specific antibody, a spike protein specific aptamer and/or an ACE2 receptor protein, a single detection binding species of a spike protein specific antibody can be used.
  • the multiplex arrays of the current invention also allow the user to gain additional important information from the patient sample by identifying non-infectious individuals.
  • This is enabled by additional steps for identifying a coronavirus internal viral protein in an in vitro sample, said steps comprising (a)(i) contacting the sample with one or more of the following capture binding species; an antibody specific to an internal viral protein and an aptamer specific to an internal viral protein (b)(i) contacting any viral protein bound to the capture species with a detection binding species selected from an antibody specific to an internal viral protein and an aptamer specific to an internal viral protein and (c) (i) detecting or determining the presence or amount of coronavirus internal viral protein.
  • the detection/measurement of an internal viral protein which is not accessible on the viral surface in combination with previous methods to identify intact virion will aid in the identification of non-infectious individuals.
  • the temporal dynamics of viral shedding and transmissibility of SARS-CoV- 2 are still being refined as further research is carried out, however the methods of the current invention can provide vital information which can be integrated with this developing knowledge and used to more accurately determine an individual's risk of spreading the virus.
  • Risk management has been central to strategies to control the current pandemic, as governments looks to balance restrictive measures such as lockdowns and social distancing with minimising economic losses.
  • the current invention can have advantages over current testing methods in various scenarios.
  • internal viral protein refers to any protein which is located on the inside of the viral lipid bilayer and which would not be accessible to a binding species without the breakdown, degradation or lysis of the virion (i.e. the protein is not found on the viral surface).
  • Internal viral proteins include nucleocapsid protein (N) and non-structural viral proteins NSP1, NSP2, NSP3, NSP4, NSP5, NSP6, NSP7, NSP8, NSP9, NSP10, NSP11, NSP12, NSP13, NSP14, NSP15 and NSP16.
  • Possible internal viral protein targets include open reading frames (ORFs) ORFlab, ORF3a, ORF3b, ORF6, ORF7a, 0RF7b, ORF8, ORF9a, 0RF9b, ORFIO and 0RF14.
  • the preferred internal viral protein target of the invention is the nucleocapsid protein, even more preferably the nucleocapsid protein of a Betacoronavirus, preferably SARS-CoV-2.
  • the capture and/or detection binding species for the internal viral protein is an antibody or aptamer specific to a SARS-CoV-2 internal viral protein. More preferably the capture and/or binding species is an antibody or aptamer to SARS-CoV-2 nucleocapsid protein.
  • the methods of the current invention can also be carried out without the need for a detection binding species therefore enabling even more rapid tests.
  • Such methods comprise the steps of (a) contacting said sample with two or more of the following capture binding species; an antibody specific to a viral surface protein, an aptamer specific to a viral surface protein and a virus receptor (b) detecting or determining the presence or amount of coronavirus virion.
  • additional steps for identifying a coronavirus internal viral protein in the patient sample can also be carried out, said steps comprising (a)(i) contacting the sample with one or more of the following capture binding species; an antibody specific to an internal viral protein and an aptamer specific to an internal viral protein and (b) (i) detecting or determining the presence or amount of coronavirus internal viraLprotein.
  • Detection and/or determination for these methods not involving a detection binding species can be achieved by electrical conductance, surface plasmon resonance, Raman spectroscopy or luminescence measurements. Electrical conductance, surface plasmon resonance and Raman spectroscopy methods are label free and therefore contain fewer assay components. However, a detection binding species could still be added to such assays to increase specificity. In luminescence-based measurements the detection binding species incorporates a detectable label.
  • a further aspect of the invention is a method in which one or more capture binding species are attached to addressable locations on a planar substrate or bead.
  • the planar substrate can be any surface able to support one or more capture binding species of the invention but is preferably a biochip.
  • a biochip is a planar substrate that may be, for example, mineral or polymer based, but is preferably a ceramic chip (AI2O3, mica etc), plastic chip, glass chip or microtitre plate that optionally incorporates a further layer comprising an ink formulation, boron nitride, graphene or graphene oxide.
  • a solid-state device that may be used in the invention may be prepared by activating the surface of a suitable substrate and applying an array of capture binding species on to the discrete sites on the surface.
  • the capture binding species may be bound to the substrate via a linker.
  • the capture binding species is an antibody, it is preferred that the activated surface is reacted successively with an organosilane, a bi-functional linker and the antibody.
  • the solid-state device used in the methods of the present invention may be manufactured according to the method disclosed in, for example, GB-A-2324866 the contents of which are incorporated herein in its entirety.
  • the solid-state device can be any substrate to which capture binding species of the current invention can be attached, for example porous paper, micro-structured polymer or sintered polymer as in a lateral flow device, or a microtitre plate or beads.
  • the solid-state device used in the methods of the present invention is a biochip.
  • the biochip may be a biochip which is incorporated into the Biochip Array Technology System (BAT) available from Randox Laboratories Limited (Crumlin, UK).
  • BAT Biochip Array Technology System
  • Figure 1 illustrates possible assay formats of the current invention. In its most basic format an assay of the current invention consists of an antibody to SARS-CoV-2 spike protein RBD and an antibody to SARS-CoV-2 nucleocapsid protein bound to a planar substrate or bead, with an optional detection binding species being introduced.
  • the in vitro sample used in the methods of the current invention is a saliva sample, an exhaled breath sample, a nasopharangyeal swab sample or an oropharangyeal swab sample but is preferably either a saliva or nasopharangyeal swab sample, and most preferably a saliva sample. Less intrusive samples such as saliva are easier to collect and more amenable to patients. In the case of SARS-CoV-2, saliva samples may have a high concentration of virion when an individual is most infectious, i.e. the optimum time of identification. Upper respiratory tract samples are preferred as viral shedding is believed to be highest just before and in the few days after symptom onset.
  • the methods of the current invention would have utility in isolating virion from lower respiratory tract samples, for example sputum samples, bronchial brush or bronchoalveolar lavage (BAL) samples. Further still, the methods could also be applied to the isolation of virion from samples from virus reservoir species such as domestic animals, including cats and dogs, farmed animals including sheep, cattle, pigs and mink or wild animals including bats and rodents. Preferably the sample used does not undergo a lysis step prior to analysis as an important aspect of the current invention is the detection of intact virion. However, the current method could be used alongside, or prior to, methods to detect viral RNA which require sample lysis. The methods of the invention described herein are carried out ex vivo. For the avoidance of doubt, the term "ex vivo" has its usual meaning in the art, referring to methods that are carried out in or on a sample obtained from a subject in an artificial environment outside the body of the subject from whom the sample has previously been obtained.
  • a further embodiment of the current invention provides a chip or bead system comprising two or more of the following capture binding species; an antibody specific to a viral surface protein, an aptamer specific to a viral surface protein and a virus receptor.
  • the chip or bead system comprises an antibody capture binding species specific to the spike protein of a coronavirus virion and an ACE-2 receptor protein to the coronavirus virion and/or an aptamer to the spike protein of a coronavirus virion.
  • the chip or bead system can comprise a further capture binding species which is specific to a nucleocapsid protein of the coronavirus and is selected from an antibody or an aptamer. Such a system can aid in the identification of infectiousness of individuals who have a positive test result for SARS-Cov-2.
  • the chip array is a planar substrate which is a ceramic chip (AI2O3, mica etc) plastic chip, glass chip or microtitre plate that optionally incorporates a further layer comprising an ink formulation, boron nitride, graphene or graphene oxide and the bead system comprises beads which are fluorescent.
  • a ceramic chip AI2O3, mica etc
  • a further layer comprising an ink formulation, boron nitride, graphene or graphene oxide and the bead system comprises beads which are fluorescent.
  • a kit comprising the chip or bead system discussed above is also provided and optionally further comprises one or more detection binding species specific to a coronavirus surface protein, preferably the coronavirus spike protein.
  • the one or more detection binding species specific to the coronavirus spike protein are specific to the same or different epitopes on the coronavirus spike protein as that of the capture binding species.
  • the detection binding species specific to the coronavirus spike protein can be an antibody, a virus receptor protein or an aptamer.
  • the kit can further comprise one or more detection binding species specific to an internal coronavirus protein, preferably the coronavirus nucleocapsid protein and which is an antibody or aptamer.
  • the coronavirus to be detected by the kit is SARS-CoV-2.
  • the methods of the current invention may be used in isolation as a rapid and accurate test to determine infection status of an individual, however, they can also be integrated with current viral RNA tests such as any PCR based tests.
  • the RNA-based test can be used to confirm the result of the methods of the invention or to add further information.
  • the current methods can be used prior to carrying out a viral RNA-based test, the advantage of this would be that they can isolate the virion from the sample meaning that any nucleic acid subsequently detected is from virions rather than residual viral fragments. Such a method would also act as a sample purification method and minimise the false negatives experienced in PCR tests due to, for example, poor sample quality.
  • a method for identifying coronavirus virions in an in vitro sample comprising the steps of (a) contacting said sample with one or more of the following capture binding species; an antibody specific to a viral surface protein, an aptamer specific to a viral surface protein and a virus receptor (b) releasing any virions bound to the capture binding species (c) lysing the free (d) detecting or determining the presence of coronavirus RNA.
  • Methods for releasing bound virions, lysis reagents which could be used, and detection methods for viral RNA are well known to those skilled in the art.
  • the preferred RNA detection method would be a reverse-transcription PCR method (RT-PCR), for example real-time RT-PCR.
  • Figure 2 illustrates examples of this method.
  • Such an embodiment would comprise a method of diagnosing a viral infectivity status of an individual suspected of being infected with coronavirus using an in vitro sample of the individual comprising (a) contacting said sample with one or more of the following capture binding species; an antibody specific to a viral surface protein, an aptamer specific to a viral surface protein and a virus receptor (b) separating out virion captured from step (a) and detecting or determining the amount of virion and/or RNA within the virion (c) subjecting the remaining sample to a PCR-based protocol to detect or determine its RNA content (d) comparing the ratio of the results obtained in steps (b) and (c) and based on the ratio assigning a coronavirus virus infectivity status to the individual.
  • references herein to the terms "infectiousness”, “infectivity status”, “actively contagious” and “transmissibility” all refer to the potential of an individual to spread a virus to others, which in the context of the current methods is gauged by detection of virion, and optionally an internal viral protein, in a sample from said individual.
  • SARS-CoV-2 RNA positive patient samples confirmed by RT-PCR, obtained from oropharangyeal and nasopharangyeal swabs were subject to the isolation procedure.
  • Anti-spike antibodies anti-RBD
  • anti-nucleocapsid antibodies were coupled to tosyl activated magnetic particles (Dynabeads M280) as follows: sodium phosphate buffer (IM, pH 8) was added to the beads in a sample tube, subjected to a magnet and the supernatant removed.
  • Ammonium sulphate (3M) and sodium phosphate buffer (pH 8) buffer, ammonium sulphate and antibody were added to the beads and incubated at 37-C for 21 hours, followed by washing in tris-buffered saline (pH 8) and 0.5% bovine serum albumin (BSA) and incubation at 37 e C for 21 hours, then washed in tris-buffered saline and tween-20 (TBST), 0.1% BSA and sodium azide (pH 7.4).
  • BSA bovine serum albumin
  • TBST tris-buffered saline and tween-20
  • Patient sample was extracted from swab and then incubated for 30 minutes at room temperature with the conjugated beads. The beads were washed in TBST buffer and magnetically separated ( Figure 1).
  • the anti-nucleocapsid beads were immediately subjected to RNA amplification using RT-PCR of ORfl primers.
  • the anti-spike beads supporting the SARS-CoV-2 virion were subject to lysis using a protein kinase/guanidinium chloride-based buffer prior to RNA amplification using RT-PCR.
  • Results from the two patient samples show that anti-spike antibodies bound to the intact virion surface spike protein and the anti-nucleocapsid antibodies to nucleocapsid protein present on swab samples from the upper-respiratory tract of SARS-CoV-2 positive patients thus allowing virion and nucleocapsid protein separation from patient sample.

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Abstract

La présente invention concerne de nouveaux procédés pour isoler et identifier des virions de coronavirus dans un échantillon qui sont indicatifs d'une infection active, mais également, de façon importante, indiquent des individus susceptibles d'être infectieux pour d'autres. Les procédés décrits ici peuvent être utilisés dans l'isolation sous la forme d'un test rapide ou peuvent être intégrés à des tests de détection d'ARN viral actuels et les améliorer.
PCT/EP2021/075660 2020-09-18 2021-09-17 Procédés pour déterminer l'état d'infectivité du coronavirus WO2022058526A1 (fr)

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CN114814216A (zh) * 2022-06-30 2022-07-29 中国科学院基础医学与肿瘤研究所(筹) 用于鉴别新冠核衣壳蛋白的核酸适体-抗体混合夹心法

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CN113786479A (zh) * 2021-01-12 2021-12-14 赛元生物科技(杭州)有限公司 一种新冠肺炎检查方法

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