WO2022129443A1 - Tests par écoulement latéral - Google Patents

Tests par écoulement latéral Download PDF

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Publication number
WO2022129443A1
WO2022129443A1 PCT/EP2021/086355 EP2021086355W WO2022129443A1 WO 2022129443 A1 WO2022129443 A1 WO 2022129443A1 EP 2021086355 W EP2021086355 W EP 2021086355W WO 2022129443 A1 WO2022129443 A1 WO 2022129443A1
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WIPO (PCT)
Prior art keywords
antibody
lateral flow
tag
protein
test strip
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PCT/EP2021/086355
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English (en)
Inventor
Peter Laing
Richard Campbell
Eric Wagner
Christopher Littlewood
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Coronex Limited
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Publication of WO2022129443A1 publication Critical patent/WO2022129443A1/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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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

Definitions

  • the invention relates to lateral flow (LF) test strips and devices comprising antigenic peptides and I or proteins for use in detection of antibody in a biological sample.
  • the LF test devices can be used for detection of infections or autoimmune disease; in particular, the LF test strips and devices can be used for specific detection of anti-Coronavirus antibody, diagnosis of Coronavirus infection and investigation of exposure to Coronaviruses.
  • Lateral flow tests for serology are immuno-chromatographic tests based on the arrest and concentration of coloured particles from an aqueous medium flowing by capillary action, usually as a line or dot on a cellulose or nitrocellulose medium that generate a visible signal in the test as a coloured signal.
  • To detect antibodies against an antigen of interest by lateral flow e.g., to assist the diagnosis of an infectious disease or a state of immunity thereto, it is usual to coat the particles themselves with the antigen, or in some designs the antigen is laid down and dried on the test strip, which is initially invisible, but which accumulates coloured particles coated in anti-immunoglobulin antibodies of appropriate class and species specificity during the running of the test, provided that antigen-specific antibodies are present in the sample.
  • the antigen has to be coated to a surface, which usually involves some degree of compromise to the structure of the antigen. While some antigens survive surface attachment well, others do not.
  • nitrocellulose is quite a denaturing surface for antigens coated directly upon it by hydrophobic interaction.
  • the chemical (e.g., amine) or surface (e.g., hydrophobic) functionality that is used for surface deposition may obscure important epitopes of the antigen. Surface denaturation, or obscuration, can result in a loss of specific signal. Specificity and sensitivity of the LF assay depends on epitope specificity of capture and detector antibodies and efficiency of conjugation of detector molecules with the coloured particles.
  • Coronaviruses are enveloped positive (+) sense, single-stranded RNA viruses that belong to the subfamily Coronaviridae, family Coronaviridae, order Nidovirales.
  • Four genera of CoVs have been identified: Alphacoronavirus (aCoV), Betacoronavirus (PCOV), Deltacoronavirus (SCoV), and Gammacoronavirus (yCoV).
  • Coronaviruses are of zoonotic origin with aCoV and pCoV being found in bats and rodents, while SCoV and yCoV are found in avian species.
  • SARS CoV-2 virus is a betacoronavirus, discovered in Wuhan City, Hubei province, China in December 2019 that can cause severe respiratory infection in humans.
  • SARS CoV-2 contains four structural proteins: envelope (E), spike (S), membrane (M), and nucleocapsid (N).
  • the S, M, and E proteins form the envelope of the virus.
  • the M protein is the most abundant and largely responsible for the shape of the envelope.
  • the E protein is the smallest structural protein.
  • the S and M proteins are also the transmembrane proteins involved in virus assembly during replication.
  • the N proteins remain associated with the RNA forming a nucleocapsid inside the envelope.
  • the N protein is involved in processes relating to the viral genome and it is also involved in other aspects of the CoV replication cycle (assembly and budding) and the host cellular response to viral infection. Polymers of S proteins remain embedded in the envelope giving it a crown-like appearance, hence the name coronavirus.
  • the Spike protein conformation of SARS-CoV-2 is comprised of S1 and S2 subunits.
  • the S1 subunit contains a signal peptide, followed by an N-terminal domain and receptor-binding domain (RBD).
  • the S2 subunit contains conserved fusion peptide, heptad repeat 1 and 2, a transmembrane domain and a cytoplasmic domain.
  • the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs and human SARS-CoV-1.
  • the S1 subunit shares 70% identity to these CoVs, but the core receptor binding domain is highly conserved. These amino acid differences are responsible for the direct interaction of spike protein with the host receptor.
  • Spike glycoprotein binds to the human ACE2 receptor present in the target cells in the respiratory tract. After the spike protein binds with the receptor in the target cell, the viral envelope fuses with the cell membrane and releases the viral genome into the target cell.
  • SARS-CoV-2 is closer to the SARS-like bat CoVs in terms of the whole genome sequence.
  • mutations observed in non-structural proteins NSP2 and NSP3 and the spike protein play a significant role in infectious capability of SARS-CoV-2.
  • SARS-CoV-2 (“L strain”) in December 2019, the S, V, G, GR and GH variants of SARS-CoV- 2 have been identified.
  • a point-of-care diagnostic test will allow identification of persons who have previously been infected, whether or not said infection was symptomatic. Such tests will help define which subjects should receive vaccination, maximising the safety and effectiveness of vaccine deployment.
  • WO2018/215495 describes lateral flow tests for the detection of flavivirus infection using modified, non-native recombinantly expressed glycoproteins, derived from the E-protein fusion loop of dengue and I or Zika flaviviruses, into which glycosylation sites were introduced and glycosylated on expression to mask the immunodominant and cross-reactive fusion-loop epitope of the E-protein.
  • the invention provides:
  • a lateral flow test strip comprising:
  • sample pad for application of a liquid sample suspected of comprising antibody to a target, said sample pad comprising:
  • one or more tagged antigenic peptide(s), comprising a recombinant or synthetic antigenic peptide of a target and an affinity-binding tag and I or
  • one or more tagged antigenic protein(s) comprising a recombinant antigenic protein (e.g., domain, or a part thereof) of the target and an affinity-binding tag, said one or more tagged antigenic peptide and I or one or more tagged antigenic protein comprising a native amino acid sequence of the target, wherein said target is selected from an infectious disease agent, an autoimmune marker or a target for a therapeutic antibody, wherein said infectious disease agent is not a Flavivirus;
  • a conjugate pad comprising a detector conjugate for labelling immune complexes formed between antibody to the target, if present in the liquid sample, and tagged antigenic peptide and I or tagged antigenic protein of the target
  • a capture strip e.g. a nitrocellulose strip
  • a capture means to capture and immobilise detector-conjugate-labelled immune complexes (detector-conjugate-labelled- antibody-tagged-antigenic peptide complexes and I or detector-conjugate-labelled-antibody- tagged antigenic protein complexes) via the affinity-binding tag, and
  • a lateral flow test strip according to clause 1 wherein the sample pad is configured or composed of material of decreasing pore size in the direction of flow to filter out blood cells and I or wherein the sample pad comprises a blood cell arresting agent.
  • 12 to 30 amino acids in length more preferably in the range of from 14 to 25 amino acids in length, yet more preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.
  • a lateral flow test strip wherein the one or more antigenic peptide and antigenic protein are derived from same target, preferably the one or more peptide and protein are derived from different protein domains of a protein, or different proteins, of the target.
  • a lateral flow test strip wherein the infectious agent is selected from the group consisting of a Coronavirus, (e.g., SARS-CoV-2), HIV, John Cunningham (JC virus, human polyomavirus 2), HSV virus and malaria;
  • the autoimmune target is selected from NMDA (N-methyl-D-aspartate) receptors, LGI1 (leucine rich glioma inactivated 1), contactin-associated protein 2 (CASPR2), AChR (acetylcholine receptor), muscle-specific kinase (MuSK), a GABA A receptor, a GABA B receptor, and a glycine receptor;
  • the therapeutic antibody target is selected from the target of natalizumab (a4- integrin), rituximab (CD20), tocilizumab (IL-6), adalimumab (TNF-a), bevacizumab (VEGF-A), infliximab (TNF-a), peripheral
  • a lateral flow test strip according to any preceding clause, wherein the infectious agent is SARS-CoV-2 and the amino acid sequence of the one or more antigenic peptide is selected from E20 (SEQ ID NO: 8) and G19 (SEQ ID NO: 13).
  • the affinity-binding tag is selected from a His tag (e.g., a Histidine multimer tag, such as 6His (hexa-His) or 5His (penta-His)), a FLAG tag, and a tag recognized by streptavidin, Avidin or NeutrAvidin (such as StrepTag-ll).
  • a His tag e.g., a Histidine multimer tag, such as 6His (hexa-His) or 5His (penta-His)
  • FLAG tag e.g., a Histidine multimer tag, such as 6His (hexa-His) or 5His (penta-His)
  • streptavidin e.g., a Histidine multimer tag, such as 6His (hexa-His) or 5His (penta-His)
  • a FLAG tag e.g., a Histidine multimer tag, such as 6His
  • a lateral flow test strip according to any preceding clause, wherein the one or more peptide antigen and tag are separated by a spacer.
  • the spacer comprises a spacer selected from a PEG spacer (e.g., a PEG-6, PEG-3, or PEG-12 spacer), an oligoglycine spacer (e.g. from two to six glycine residues) and an amino acid sequence of 4 to 6 amino acid residues.
  • a PEG spacer e.g., a PEG-6, PEG-3, or PEG-12 spacer
  • an oligoglycine spacer e.g. from two to six glycine residues
  • amino acid sequence 4 to 6 amino acid residues.
  • a lateral flow test strip according to any preceding clause wherein the detector conjugate is coloured particles (e.g., colloidal gold nanoparticles) or fluorescent particles conjugated to an anti-species Ig antibody, e.g., anti-human Ig antibody. 12. A lateral flow test strip according to any preceding clause, wherein the detector conjugate is coloured colloidal gold nanoparticles conjugated to an anti-human Ig antibody selected from anti-human IgG antibody, anti-human IgA antibody, anti-human IgM antibody, or a mixture of any 2 or all 3 thereof.
  • the detector conjugate is coloured particles (e.g., colloidal gold nanoparticles) or fluorescent particles conjugated to an anti-species Ig antibody, e.g., anti-human Ig antibody. 12.
  • the detector conjugate is coloured colloidal gold nanoparticles conjugated to an anti-human Ig antibody selected from anti-human IgG antibody, anti-human IgA antibody, anti-human IgM antibody, or a mixture of any 2
  • a lateral flow test strip according to any preceding clause, wherein the affinity-binding tag is a His multimer tag (e.g., a 6His tag (SEQ ID NO: 2)) and the capture means for capture of the detector-conjugate-labelled immune complexes comprises an antibody to the His multimer tag.
  • the affinity-binding tag is a His multimer tag (e.g., a 6His tag (SEQ ID NO: 2)) and the capture means for capture of the detector-conjugate-labelled immune complexes comprises an antibody to the His multimer tag.
  • a lateral flow test strip according to any preceding clause, wherein the capture means is provided as a line or dot on the capture strip.
  • a lateral flow test strip further comprising one or more positive control of species Ig, e.g., human Ig, e.g., a positive control of human IgG, human Ig M and I or human IgA on the capture strip, e.g. provided as a control line or control dot on the capture strip.
  • species Ig e.g., human Ig, e.g., a positive control of human IgG, human Ig M and I or human IgA on the capture strip, e.g. provided as a control line or control dot on the capture strip.
  • a lateral flow test strip according to any preceding clause, wherein the liquid sample comprises a biological sample.
  • liquid sample comprises a biological sample selected from blood, plasma, serum, saliva, oral fluid, urine, faeces and CSF.
  • a diagnostic test device comprising a lateral flow test strip according to any one of the preceding clauses, said lateral flow test strip being housed within a casing, said casing comprising (a) a window or windows for inspection (e.g. visual inspection) of the test result(s) and, if present, control (s) , and
  • a diagnostic test device wherein during use the accumulation of coloured or fluorescent particles produces a colour or signal (e.g., as a line or dot on the test strip) indicative of the presence in the liquid sample of an antibody specific for the infectious disease agent, autoimmune marker or therapeutic antibody target.
  • a colour or signal e.g., as a line or dot on the test strip
  • a diagnostic test device, lateral flow test strip, tagged antigenic peptide and I or tagged antigenic protein according to any preceding clause for use in an in vitro method for specific detection of antibody specific for an infectious agent, diagnosis of infection and I or to investigate exposure to an infectious agent; or specific detection of antibody specific for an autoimmune marker or therapeutic antibody target.
  • a diagnostic test device, lateral flow test strip, tagged antigenic peptide and I or tagged antigenic protein according to any preceding clause for use in an in vitro method for specific detection of anti-SARS-CoV-2 antibody, diagnosis of SARS-CoV-2 infection and I or to investigate exposure to SARS-CoV-2.
  • a diagnostic test kit comprising a lateral flow test strip, diagnostic test device, tagged antigenic peptide and I or tagged antigenic protein according to any preceding clause, and one or more reagent(s) and I or instructions for use.
  • a method for detection of an antibody e.g. an anti-SARS-CoV-2 antibody
  • a lateral flow test strip comprising use of a lateral flow test strip, diagnostic test device, tagged antigenic peptide and I or tagged antigenic protein, or kit according to any preceding clause.
  • tagged antigen is not bound either covalently or non-covalently to a surface, instead, upon sample and diluent application to the lateral flow test strip, tagged antigen is free to react with antibodies, if present, in the test sample in the aqueous phase of the test; the structure of epitope of the antigen is not compromised by surface absorption or obscuration.
  • the coloured particles of the test are concentrated and arrested at the test line or dot via a capture means, preferably an anti-tag antibody that recognises specifically a tag on the antigen. This design allows the antiimmunoglobulin antibody of the test (instead of the antigen) to be attached to the coloured particle.
  • Antibodies such as monoclonal anti-immunoglobulin antibodies are generally more tolerant to surface attachment, possibly because their multi-domain structure allows deposition of non-antigen-binding domains to the surface in question, without significant compromise to their antigen-specific binding properties. Furthermore, the use of an anti-tag antibody for arrest guards against prozone effects that would otherwise obtain if an epitope of the antigen and a corresponding anti-epitope antibody were used to effect particle arrest. (In such scenario, i.e. , use of an epitope-specific antibody as distinct from an anti-tag antibody, antibodies of the sample that recognize said epitope would prevent signal development even in the presence of antigen-specific antibodies in the test sample).
  • the strip design comprises the overlapping arrangement in sequential order of (a) sample pad, (b) conjugate pad, (c) capture strip (e.g., nitrocellulose membrane) and (d) absorbent pad.
  • a lateral flow immunoassay (LFIA) test strip comprises these elements sequentially arranged as a strip on a backing material such as a plastic I adhesive backing.
  • the test strip is placed in specially-designed cassette or case to provide a diagnostic test device, this helps to ensure proper flow rate and stability.
  • the Sample pad may be composed of cellulose, cross-linked silica or glass fibres and acts as the platform for sample analyte. Glass fibre is often used as the sample pad due to its low protein affinity and high absorption capacity, providing a steady and uniform flow of sample to conjugate pad.
  • the sample pad is the main site of pre-treatment in LFIA for reducing background noise and non-specific interactions.
  • the sample pad can be configured or composed of material for blood cell separation and background clearing.
  • the sample pad may be configured or composed of material, e.g. such as cellulose acetate, of decreasing pore size in the direction of flow, designed to filter out blood cells additionally or alternatively the sample pad may comprise a blood cell arresting agent, such as an anti-glycophorin antibody.
  • cell separating membranes may be used in the sample pad in continuation and overlap with the conjugate pad.
  • the sample pad comprises (i) one or more tagged antigenic peptide, comprising a recombinant or synthetic antigenic peptide of a target having an affinity-binding tag and I or (ii) one or more tagged antigenic protein, comprising a recombinant antigenic protein (e.g., domain) of the target having an affinity-binding tag.
  • the tagged antigenic peptide and I or tagged antigenic protein comprise a native amino acid sequence of the target that is capable of forming an epitope bound by antibody to the target, if present, in the liquid sample.
  • the (i) one or more tagged antigenic peptide, comprising a recombinant or synthetic antigenic peptide of a target having an affinity-binding tag and I or (ii) one or more tagged antigenic protein, comprising a recombinant antigenic protein (e.g., domain) of the target having an affinity-binding tag are not comprised in the sample pad and instead may be combined with the liquid sample before application to the sample pad.
  • the one or more antigenic peptide and I or one or more antigenic protein are from the same target.
  • the one or more antigenic peptide and I or one or more antigenic protein are from different protein domains of a protein of the target, in some embodiments the one or more antigenic peptide and I or one or more antigenic protein are from different proteins of the target.
  • the one or more antigenic peptide is in the range of from 8 to 50 amino acids in length, preferably in the range of from 12 to 30 amino acids in length, more preferably in the range of from 14 to 25 amino acids in length, yet more preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.
  • the one or more antigenic protein is greater than 50 amino acids in length.
  • the target is selected from an infectious disease agent, an autoimmune marker or a target for a therapeutic antibody.
  • the modified tagged peptide sequences are designed for diagnostic use, as antigens for detection of antibodies to a specific infectious agent, autoimmune target or target of a therapeutic antibody.
  • any immunodominant epitope of an infectious agent, autoimmune antigen or therapeutic antibody that is comprised of a short contiguous peptide sequence would be amenable to use in this lateral flow design.
  • the specific short immunodominant sequence can be chemically synthesised and tagged as described and then used within the described architecture to form a new assay for the detection of antibodies against the epitope.
  • pathogenic antibodies against the N-methy-D-aspartate (NMDA) receptor cause a severe form of autoimmune encephalitis: anti-NMDA receptor encephalitis.
  • the vast majority of pathogenic antibodies in patients recognise a short amino acid sequence within the GluN1 amino terminal domain.
  • Current diagnostic assays comprise of cell-based assays in which transfected cDNA results in expression of whole NMDA receptor protein on the cell surface. This then serves as a substrate for detecting antibody binding.
  • the lateral flow design described herein can be used by chemically synthesizing the necessary and sufficient short peptide sequence of the amino terminal domain and modifying with the aforementioned tagging to generate a unique lateral flow test for detecting pathogenic anti- NMDA receptor antibodies.
  • a unique, short, contiguous peptide sequence of a therapeutic antibody target can be chemically synthesised and tagged to form a unique assay for the measurement of that particular therapeutic antibody.
  • rituximab is a widely- used chimeric (mouse constant region; human variable region) therapeutic antibody that binds to CD20 expressing B-cells and destroys them.
  • a short, contiguous, peptide sequence of the epitope that rituximab recognises can be chemically synthesised as described and used in a unique lateral flow assay to detect the presence of the therapeutic antibody.
  • the target is an infectious agent it may be selected from the group consisting of a Coronavirus, (e.g., SARS-CoV-2), HIV, John Cunningham (JC virus, human polyomavirus 2), HSV virus and malaria.
  • a Coronavirus e.g., SARS-CoV-2
  • HIV e.g., SARS-CoV-2
  • JC virus e.g., human polyomavirus 2
  • HSV virus e.g., human polyomavirus 2
  • the target is an autoimmune target it may be selected from NMDA (N-methyl-D-aspartate) receptors, LGI1 (leucine rich glioma inactivated 1), contactin-associated protein 2 (CASPR2), AChR (acetylcholine receptor), muscle-specific kinase (MuSK), a GABA A receptor, a GABA B receptor, and a glycine receptor.
  • NMDA N-methyl-D-aspartate
  • LGI1 leucine rich glioma inactivated 1
  • CASPR2 contactin-associated protein 2
  • AChR acetylcholine receptor
  • MusSK muscle-specific kinase
  • target is the target of a therapeutic antibody
  • the target may be selected from the target of natalizumab (a4-integrin), rituximab (CD20), tocilizumab (IL-6), adalimumab (TNF-a), bevacizumab (VEGF-A), infliximab (TNF-a), pembrolizumab (PD-1), trastuzumab (HER2) and alemtuzumab (CD52).
  • the target is not a Flavivirus and the tagged antigenic protein is not a wild type or modified Flavivirus E protein fusion loop protein.
  • the infectious agent is SARS-CoV-2 virus and the one or more antigenic peptide is an antigenic SARS-CoV-2 native peptide and I or the one or more antigenic protein is a SARS-CoV-2 native protein, e.g., a domain or a part thereof of a SARS CoV-2 protein.
  • the protein is a domain or part thereof of an S protein, an N protein, or an Orf3a protein from SARS-CoV-2.
  • the infectious agent is SARS-CoV-2 and the amino acid sequence of the one or more antigenic peptide is one or more peptide of the spike protein, preferably selected from E20 (SEQ ID NO: 8) and G19 (SEQ ID NO: 13) and I or and the amino acid sequence of the antigenic protein is RBD (SEQ ID NO: 1).
  • the tagged antigenic protein is RBD6His (SEQ ID NO: 3).
  • Affinity-binding tags were developed for protein detection, characterization, and purification. Affinity tags commonly used in recombinant protein production and suitable for use in the invention are described below. Particularly preferred are small tags e.g., 6 to 12 amino acids residues long to minimise the possibility that an antibody of the sample and the anti-tag antibody could bind simultaneously.
  • Polyhistidine tag, or nHis is a polypeptide consisting of several histidine residues that can be located on the N or C-terminus of a recombinant protein or peptide. nHis is a short sequence usually of five (penta-His, 5His) or six (hexa-His, 6His) histidine residues. nHis does not alter the charge of the protein and during recombinant expression does not adversely affect protein transfer and folding within the cell.
  • Polyhistidine tags have been used successfully for purification of recombinant proteins in various expression systems, including bacterial, yeast, plant cell and mammalian cells systems. The polyhistidine tag does not generally affect the structure and function of the purified protein. Highly specific monoclonal antibodies to polyhistidine tag are commercially available.
  • the affinity-binding tag is nHis, preferably 6His, and the capture means is an anti-His tag antibody.
  • Polyarginine tag typically consists of five or six consecutive arginines at the C-terminus end of a protein or peptide.
  • Poly-Arg tag is used for purification of recombinant proteins, the presence of the polyarginine tag confers a positively charge and thus with affinity for a negatively charged sorbent, so it may affect tertiary structure of protein and/or protein properties.
  • FLAG tag consisting of eight amino acids and with molecular weight of 1 kDa, is also successfully used in affinity chromatography.
  • the small size of the tag minimizes any interfering effect.
  • application of FLAG tag in low pH elution may irreversibly affect protein properties.
  • Streptavidin-binding peptide or Streptavidin-binding protein is 38 residues in length and can be used to immobilize fusion proteins on a streptavidin matrix. Recombinant proteins containing the SBP-tag bind to streptavidin and this property can be utilized in specific purification and detection.
  • Streptavidin-binding tag is a small affinity protein that is used as a sorbent. Because the affinity of biotin for streptavidin is higher than that of any of the streptavidin- binding peptides, Strep-tagged fusion proteins can be efficiently eluted by free biotin. Strep- tag is infrequently used as it is restricted by its location only on the C-end and by its relatively low affinity.
  • Strep-tag II also called modified streptavidin-binding tag, is a small affinity peptide (WSHPQFEK) that was simultaneously optimized compared with Strep-tag.
  • Strep-tag II is inert, largely resistant to cellular proteases, can be used with mild detergents. Strep-tag II works equally well on both the C- and N-ends of the recombinant protein and it is optimal for the purification of recombinant proteins under physiological conditions.
  • Twin-Strep tag consisting of two Strep-tag®ll moieties connected by a short linker, is developed to improve the binding characteristics of Strep-tag II when it is used in the protein purification from large volumes.
  • the Twin-Strep-tag features all beneficial properties of Strep- tag II, including efficient elution under gentle competitive conditions.
  • Twin-Strep-tag enables a more universal use in applications requiring stable binding because of its higher affinity.
  • Chitin-binding tag comes from Bacillus circulans and consists of 51 amino acids.
  • the recombinant protein binds with the sorbent (chitin immobilized on Sepharose) under physiological conditions.
  • CBD fusion proteins are purified by affinity chromatography on chitin resin.
  • Commercially available vectors provide for intein-CBD expression on the N-terminus, C-terminus, or both termini of a heterologous protein of interest.
  • Natural histidine affinity tag epitope is a naturally-occurring sequence of non-adjacent histidine residues that has a lower overall charge than tags with consecutive His residues.
  • HAT-protein fusions exhibit solubility that more closely resembles that of wild-type proteins, while still possessing strong affinity for immobilized metal ions.
  • HAT-tagged protein is soluble, therefore it does not form aggregates and it can be eluted under mild conditions such as low imidazole
  • the affinity-binding tag is selected from a His tag (e.g., a Histidine multimer tag, such as 6His (hexa-His, SEQ ID NO: 2) or5His (penta-His)), a FLAG tag, and a tag recognized by streptavidin.
  • a His tag e.g., a Histidine multimer tag, such as 6His (hexa-His, SEQ ID NO: 2) or5His (penta-His)
  • 6His hexa-His, SEQ ID NO: 2
  • 5His penta-His
  • the affinity binding tag may be positioned C-terminally or N-terminally of the antigenic protein and I or antigenic peptide. In specific embodiments described herein the affinity tag is positioned C-terminally to the protein antigenic and I or antigenic peptide.
  • the peptide antigen and affinity tag are separated by a spacer.
  • a spacer between the antigenic peptide and affinity tag may permit the antigenic peptide to adopt a suitable configuration to display its epitopes(s) in a native format to enable binding to an antibody of the test sample and also to allow the tag (e.g., 6His) to be displayed so that it can be captured by the capture means (e.g., anti-His-antibody).
  • the spacer may comprise a PEG spacer, such as a PEG-6, PEG-3, or PEG-12 spacer, an oligoglycine spacer (e.g. from two to six glycine residues) or an amino acid sequence, for example of 4 to 6 amino acid residues.
  • the amino acid sequence of the spacer may comprise a sequence of amino acid residues of the native sequence of the target, e.g. residues of the target that do not comprise the epitope of the antigenic peptide of the target, optionally with one or more amino acid modifications to alter one or more, but not all, amino acid residues to non-native sequence, or it may comprise a short sequence, e.g., of 4 to 6 residues, unrelated to the target sequence.
  • the spacer is a PEG-6 spacer, a diglycine spacer, or 4, 5, or 6 residues of the native sequence of the target, outside of the target epitope, optionally with 1 , 2, or 3 modifications of native residues to non-native residues, preferably at one or more C-or N- terminal residues of the antigenic peptide.
  • the conjugate pad materials include glass fibres, cellulose filters and polyester.
  • the conjugate pad comprises the detector conjugate.
  • the detector reagent is responsible for the formation of colour or fluorescent signal in LFIA due to aggregation of nanoparticles or microparticles.
  • Gold nanoparticules or coloured latex microparticles may be used as the detector reagent in the detector conjugate.
  • Gold nanoparticles are widely used for conjugation with antibodies because of their inert nature.
  • nanoparticles in the range of from 20-80 nm are used for conjugation purposes and nanoparticles in the range of from 40 to 60 nm are generally preferred.
  • the colour of the nanoparticles is dependent on their size.
  • nanoparticles are possible depending on the nature of the nanoparticle, whether it is gold, its size I shape, and/or whether it is a dyed particle such as NanoActTM from Asahi Kasei of Japan, which are intensely coloured cellulose nanoparticles).
  • Other labels suitable for use as detector reagent in the detector conjugate include upconverting phosphors, magnetic particles, carbon I graphene nanoparticles, silver nanoparticles, quantum dots, cellulose nanobeads, and silica. Chemical labels that have an intrinsic signal such as fluorescence or near-infrared may be employed with a suitable detection system for visualisation.
  • the detector conjugate (also referred to as detector antibody conjugate) comprises detector reagent (e.g., coloured or fluorescent, nanoparticles or microparticles) conjugated to an antiimmunoglobulin antibody (anti-lg antibody).
  • detector reagent e.g., coloured or fluorescent, nanoparticles or microparticles conjugated to an antiimmunoglobulin antibody (anti-lg antibody).
  • the detector conjugate comprises gold nanoparticles and an anti-lg antibody specific for an immunoglobulin of the species it is intended to detect in the test liquid sample.
  • anti-species antibody for binding to the relevant non-human species Ig may be used, for example for detection of antibody in samples from livestock or companion I pet animals.
  • anti-human-lg may be used to detect the presence of human antibodies in a sample from a non-human animal engineered to express human antibody.
  • the anti-species Ig antibody is an anti-human Ig antibody, depending of the isotype of the human immunoglobulin of interest, the anti -human Ig antibody may be selected from anti-human IgG antibody, anti-human IgA antibody, anti-human IgM antibody, or a mixture of any 2 or all 3 thereof, e.g., anti-human IgG antibody and anti-human IgM antibody, anti-human IgG antibody and anti-human IgA antibody, anti-human IgA antibody and anti-human IgM antibody, or anti-human IgG antibody, anti-human IgA antibody and antihuman IgM antibody.
  • the capture strip is typically nitrocellulose film, cellulose film, filter paper, or agarose.
  • a nitrocellulose (NC) strip is used and the capture means and optional control means, e.g., antibodies, for the test and control are coated thereon, e.g., as lines transverse to the flow of liquid or as a dot.
  • the capture means for capture of the detector-conjugate-labelled immune complexes is a binding partner specific for the affinity-binding tag.
  • the affinity binding tag is a His multimer tag, such as 6His
  • the capture means is an anti-His-tag antibody capable of binding specifically to the His tag.
  • control means is an immunoglobulin of the species under test. Binding of the anti-species-lg of the detector conjugate to the species Ig results in accumulation and a visually-detectable control signal.
  • control means is a human Ig antibody or serum. Accordingly, one or more positive control of species Ig may be provided on the capture strip, e.g., for human Ig, a positive control of human IgG, human Ig M and I or human IgA may be provided on the capture strip, e.g. as a test line or test dot on the capture strip.
  • the absorbent pad is formed of cellulose. It is placed at the end of the strip distant from the sample pad and absorbs the complete solution from membrane, providing maximum background clearing.
  • the test sample is applied in the form of a liquid sample comprising a biological sample
  • the biological sample is selected from blood, plasma, serum, saliva, oral fluid, urine, faeces and CSF; the biological sample may be combined with a diluent to form the liquid sample.
  • Diagnostic aspects of the invention are exemplified using antigenic Coronavirus peptides and I or proteins in which the natural (native, wild-type) peptide sequence is labelled with an affinity tag. Such modification enables efficient pull-down and detection of immune complexes formed between the tagged antigen and antibody present in a biological sample, such as blood (serum I plasma), at the test line of a lateral flow test.
  • test antigen(s) in the sample application pad provides time, during the running of the test, for the antibodies of the test sample to react, in solution phase, unencumbered and undistorted by surface adsorption, with the tagged antigen forming an immune complex, before it is arrested at the test line via its affinity tag, making for efficient detection of the antibodies.
  • antibody we include the meaning of a substantially intact full antibody with Fc.
  • the term antibody also includes all classes of antibodies, including IgG, IgA, IgM, IgD and IgE.
  • the term antibody includes variants, fusions and derivatives of any defined antibodies and antigen binding portions thereof.
  • nucleic acid sequence encoding an antigen of the invention can generally be expressed following the functional and operable insertion of the DNA sequence into an expression vector containing control sequences and secretory signal sequences.
  • a suitable promoter for expression of nucleic acid sequences of protein or peptide of the invention is CMV for expression in mammalian cells.
  • Host cells that may be employed to express protein or peptide of the invention include the mammalian HEK and CHO cell lines, insect cells such as Tni and Drosophila S2; yeasts and non-mycelial fungi cells.
  • the term "recombinant” refers to the use of genetic engineering methods (cloning, amplification) to produce a protein or peptide antigen for use in the present invention.
  • synthetic refers to chemically synthesized antigens such as peptide antigens, e.g., made by Merrifield solid phase peptide synthesis or other methods known in the art. Most favourably these peptides faithfully represent epitopes of the target and correspond to the wild type amino acid sequence of the target protein.
  • the term "antigen” refers to an antigenic peptide or antigenic protein.
  • a peptide antigen is typically in the range of from six to fifty amino acid residues in length and capable of being synthesized on a peptide synthesizer (e.g. an automated peptide synthesizer operating the Merrifield system of solid phase peptide synthesis).
  • Peptides for may be N- terminally blocked, e.g., by acetylation.
  • a protein is greater than 50 amino acid residues in length and typically between about 7,000 and 100,000 Daltons.
  • the term "immunogenic” or “antigenic” relates to being capable of generating an immune response in vivo such as an antibody response of IgG, IgM, IgA, IgE class or any subclasses therein, or of generating T-cell responses such as antigen-specific cytokine secreting T-cells (e.g. CD4 positive antigen specific T-cells secreting interferon-gamma). Likewise it also refers to generating a cytotoxic T-lymphocyte response, i.e. , a CTL response.
  • immunogenic or “antigenic” encompasses peptides or proteins capable of forming an epitope that can be recognised by an antibody, if present, in a sample.
  • Mutation is used to describe a replacement of a base, or a deletion or insertion event in the nucleic acid encoding a protein. In some instances mutation will give rise to a change in the amino acid sequence of the protein encoded by the nucleic acid, providing a mutant or variant protein.
  • viral variant mutations that give rise to new nucleic acid or protein sequences are termed “viral variant”, thus herein “viral variant” or “variant” refers to a virus which may have one or a combination of different mutations when compared to a reference or wild type strain. Wild type and variant sequences that occur naturally, i.e. that are found in viral isolates from patients, are termed “native” sequences herein. Generally the first viral isolate L form of SARS-CoV-2 is a suitable reference strain.
  • Tests of the invention are ‘serologically’ based, detecting the presence of antibodies, e.g., in preferred embodiments to determine whether a subject has previously had SARS-Cov-2 Coronavirus infection, because the infection and vaccination history of a subject is an important determinant of how they will respond to new vaccines when licensed and in development, and may determine a subject’s susceptibility to adverse responses to vaccination. Diagnostic tests of the invention can be used to identify prior SARS-CoV-2 Coronavirus infection by detecting antibodies in a finger prick blood sample.
  • Diagnostic tests of the invention are provided preferably in lateral flow format for detection of antibodies.
  • diagnostic tests of the invention are provided in lateral flow format for detection of antibodies against SARS-CoV-2.
  • diagnostic tests of the invention comprising tagged antigenic peptides and I or tagged antigenic proteins of the invention are provided in ELISA format for detection of antibodies, e.g. antibodies against SARS-CoV-2.
  • the tests will help define whether a subject has responded adequately to a vaccine (e.g., reached a 'to be determined' protective level of anti- SARS-CoV-2 antibodies) or whether they may require further immunisation.
  • SARS-CoV-1 i.e., ‘SARS’
  • SARS-CoV-2 which is in excess of 70% identical in sequence to SARS-CoV-2 (responsible for COVID-19)
  • seasonal coronaviruses such as NL63 and HKU.
  • SARS-CoV-1 i.e., ‘SARS’
  • SARS-CoV-2 which is in excess of 70% identical in sequence to SARS-CoV-2 (responsible for COVID-19
  • NL63 and HKU seasonal coronaviruses
  • the diagnostic tests of the invention may play an important role in monitoring SARS-CoV-2 infection and distinguishing it from other cli nically-simi lar infections.
  • a 'point-of-care' diagnostic test of the invention i.e., a test that can be run without the need for clean water, electricity or equipment, which can be operated in the home or in a vaccination clinic, which can identify the presence of antibodies to SARS- CoV-2.
  • the test device is provided as a lateral flow device.
  • the diagnostic tests of the invention are enabled by the design of antigens with affinity tag.
  • a composition comprising a tagged antigenic peptide and I or tagged antigenic protein for use in the invention and a diluent.
  • a composition may comprise one or more Coronavirus antigens.
  • a composition may comprise one or more, e.g., one, two, three, four, five, six, seven, eight, nine or ten antigens of the invention, e.g., one or more different spike protein antigens, NP antigens, or a mixture thereof.
  • the Coronavirus is a SARS-CoV-2 virus and the antigenic protein is RBD (SEQ ID NO: 1 and I or the antigenic peptides are selected from E20 (SEQ ID NO: 8) and G19 (SEQ ID NO: 13).
  • the invention provides an LF test strip or device of the invention for use as a diagnostic device.
  • the invention provides a diagnostic kit comprising a LF test strip or device of the invention and a specimen diluent.
  • the invention provides lateral flow test strips and devices for use to assess if an individual is seronegative and thus has not been exposed to SARS-CoV-2, or if an individual is seropositive and has been exposed to SARS-CoV-2.
  • the invention provides diagnostic approaches that can be used for selection of subjects for immunisation, or for assessment of seroconversion to determine if immunisation has raised a sufficiently-protective immune response against SARS-CoV-2.
  • the invention provides diagnostic approaches that enable interrogation of the immune response to SARS-CoV-2.
  • the disclosure provides a LF diagnostic device of the invention for use in an in vitro method for diagnosis of SARS-CoV-2 infection and/or to investigate exposure to SARS-CoV-2 , to determine if a subject proposed for immunisation is naive to SARS-CoV-2 infection and I or has been exposed to SARS-CoV-2 infection, thereby to inform the decision to immunise against SARS-CoV-2 if the subject is naive to SARS-CoV-2 infection, or not to immunise if prior exposure to SARS-CoV-2 is detected.
  • lateral flow devices that can be used to detect exposure to SARS-CoV-2 (and can differentiate between Coronaviruses) using tagged SARS- CoV-2 peptide and I or protein antigens in a lateral flow format suitable for point-of-care diagnostic use.
  • FIG. 1 Lateral flow device design: (1) Sample Pad (with dried His-tagged SARS-CoV-2 RBD antigen, site of single application port for sample & diluent, (2) Conjugate Pad (with dried Au-MCAB anti-IgG-Fc), (3) capture strip (e.g., nitrocellulose) (4) T (test) line (e.g., MCAB anti- His-tag), (5) C (positive control) line of human IgG, (6) backing card, (7) position of absorbent pad (not shown), (8) direction of flow along strip, (9) uncaptured (off-target) materials from the test sample are carried invisibly past the observation window.
  • Sample Pad with dried His-tagged SARS-CoV-2 RBD antigen, site of single application port for sample & diluent
  • Conjugate Pad with dried Au-MCAB anti-IgG-Fc
  • capture strip e.g., nitrocellulose
  • T (test) line e.g., MCAB anti- His-tag
  • Example 1 Lateral flow tests to detect antibody in SARS-CoV-2 PCR-confirmed- positive or pre-COVID-19-negative sera
  • Pre-SARS-CoV-2-era control sera were obtained from the Glasgow Neuroimmunology Biobank (REC reference 11/WS/0025) and comprise material surplus to routine clinical use and used with permission for research and development purposes without further consent as permitted under the NHS Greater Glasgow and Clyde biorepository governance policy.
  • the ACE2 receptor-binding domain ‘RBD’ of SARS-CoV-2 spike protein (PMID 32225176) comprising residues R319-F541 (SEQ ID NO: 1) (of reference sequence YP_009724390.1 , which is ‘REFSEQ: accession NC_045512.2’, also identical to that of the protein sequence of the peptide array) was made by transient plasmid transfection of human embryonic kidney cells (HEK-293) according to Stadlbauer et al. PMID: 32302069.
  • the RBD protein was made from a plasmid with open reading frame created by de novo gene synthesis (Genewiz).
  • a Hexa-His (HHHHHH SEQ ID NO: 2) tag was appended to the C-terminal end of the RBD comprising residues R319-F541 (SEQ ID NO: 1) to form the His-tagged RBD antigen sequence in the encoding plasmid in order to allow purification of the expressed His-tagged RBD antigen protein, RBD-6His (SEQ ID NO: 3) by nickel-chelate affinity chromatography, and also to allow arrest or anchorage of the antigen in native form (without obscuration of any of its epitopes) in lateral flow and ELISA tests, via an anti-His-tag monoclonal antibody capable of recognizing C-terminally-His6-tagged RBD antigen protein RBD-6His (SEQ ID NO: 3).
  • a further advantage of the His tag exploited in this way is that it is not essential to purify or isolate the antigen protein, which can be used as supernatant in various diagnostic tests that employ the His tag for recognition or arrest.
  • RBD-6His Protein (SEQ ID NO: 3) was expressed recombinantly in human embryonic kidney cells (HEK 293) by transient transfection using the Expi 293 transfection kit (Thermo) and purified by immobilized metal affinity chromatography using Nickel NTa resin (Qiagen).
  • Expi 293 cells were routinely maintained at between 3-5e6 cells/mL in Expi293 media - grown at 37C, 8% CO2, 125rpm. 25mL of cells at 3e6/mL were transfected (Expi293 transfection kit) with 25pg of the pCACGG expression plasmid containing the recombination RBD sequence. 18-22 hours post transfection, enhancer (Expi293 transfection kit) was added to the cells, which were then harvested 3 days later for purification.
  • the cell supernatant was harvested by centrifugation then cleared through a 0.2pm filter.
  • the protein was purified on NiNTa agarose using 50mM sodium phosphate pH 7.5 based buffers. The supernatant was incubated with 0.4m L of resin for 2 hours at room temperature and eluted following incubation for 1 hour at room temperature in 3 column volumes (1.2mL) of buffer containing 235mM imidazole. The eluted protein was dialysed into DPBS. A Bradford assay and SDS PAGE were performed, according to the manufacturer’s instructions, to analyse the protein.
  • RBD-6His antigenic protein The antigen used was as follows: in ‘direct’ formats, RBD-6His antigen (SEQ ID NO: 3) was applied directly to the nitrocellulose strip in Oxoid-PBS without additives and dried; in ‘indirect’ formats, where antigen was applied to the sample application pad, the antigen was applied in Oxoid-PBS with 2% w/v sucrose and dried.
  • RBD LF test components (For “TBC” see Table 6 conditions set “B”).
  • the antigen is preferably tagged receptor binding domain RBD-6His (SEQ ID NO: 3), which is dried down in the sample application pad of the test.
  • signal is generated if there are antibodies present against the RBD (SEQ ID NO: 1) in the test sample. Lateral flow tests to detect antibody in serum pools of SARS-CoV-2 PCR-confirmed- positive or pre-COVID-19-negative sera.
  • Negative Sera Table 5 Negative Sera ( Figure 7)
  • RBD6His (SEQ ID NO: 3) in PBS sucrose (2% w/v), control line human IgG 1.0 mg/ml (deposited at 1 mg/ml, deposition rate 0.1 pl/mm, 4mm strip width), per test.
  • the optimal conditions of B may be substituted into Table 1 above, for ‘TBC’.
  • the components of a preferred embodiment of the RBD LF test are shown in Table 7 below. Note, the use of differing murine IgG subclasses for the anti-human-IgG and the anti-His tag, is favoured, since this insulates against false positives resulting from murine-IgG-directed heterophile antibodies.
  • an IgM-class antibody may be used as one of the antibodies (e.g. murine anti-human-IgG from East Coast Bio, cat no. HM295).
  • RBD receptor binding domain of SARS-CoV-2 having a hexahistidine C-terminal tag (SEQ ID NO: 3) to enable arrest by the anti-His-tag antibody.
  • the examples and figures illustrate the utility of tagged RBD protein antigen in serodiagnosis of Sars-Cov-2 in lateral flow format.
  • Human IgG antibodies are detected by virtue of a colloidal gold conjugate with surface bound anti-human-IgG.
  • an anti-tag antibody in this example a monoclonal anti-His-tag antibody was used.
  • Human IgG antibodies are detected by virtue of a colloidal gold conjugate with surface bound anti-human-IgG.
  • other antibody classes and subclasses of human and other species can be detected in this way (e.g. IgM, IgA and subclasses of IgG) by use of appropriate species and isotype specific antibodies on the colloidal gold reagent, and likewise by use of corresponding pure antibodies comprising the control line (i.e. IgM for an IgM test, IgA for an IgA test, IgG for an IgG subclass-1 for an IgG subclass-1 test etc.).
  • the detector reagent e.g., colloidal gold
  • an anti-human- IgM mu-chain specific antibody preferably a monoclonal IgM antibody, e.g. an IgG class antihuman mu-chain antibody, most preferably an IgM-class anti-human mu-chain antibody.
  • an additional advantage of exploiting anti-tag antibodies for indirect antigen-capture in lateral flow tests is that the test is made more economic with respect to antigen costs in manufacturing.
  • the economic advantage of this approach is further enhanced by the stronger recognition of indirectly-immobilised antigen (via, e.g., anti-tag antibody), compared to that deposited directly on the lateral flow capture strip (e.g. a nitrocellulose strip).
  • a further feature is that immobilisation of antigen via its affinity-tag permits functional display of neutralising epitopes supporting the use of the antigens in serodiagnostic tests, in particular point-of-care serodiagnostic tests, for the detection of neutralising antibodies.
  • Antibodies which dominate the antibody response may be poorly neutralising and their concentration may rapidly decline.
  • the lateral flow test strips and devices described here can be used to assess vaccine performance, e.g. to determine whether an additional dose may be needed, or how well a vaccine is performing in a subject already exposed to another Coronavirus by reason of infection or immunisation.
  • the lateral flow test strip comprises an adhesive backing card onto which the various components are attached.
  • the sample was applied to the sample pad port where it dissolved the His-6-tagged antigen which had been dried into the sample pad.
  • antibodies from the sample bound to the tagged antigen and formed an immune complex, which was arrested at the test line via the His tag of the antigen binding to an anti-His antibody immobilised on the test line.
  • the liquid sample hydrated the dried detector conjugate (nanoparticulate colloidal gold coated (conjugated) with an anti-hlgG antibody) provided in the conjugate pad.
  • the solution phase was more conducive to antigen-antibody reaction than solid phase reactions due to the diffusibility of reactants.
  • Peptide Arrays CelluspotTM Peptide arrays on microscope slides, representing the entire amino-acid sequence of the SARS-CoV-2 spike glycoprotein QHD43416.1 (COVID19_HullB Celluspot) were provided by Intavis AG of Germany, comprising overlapping 15-mer peptides with a 5-residue shift from spot to spot covalently attached via their C-termini to a cellulose matrix. Peptides were N-terminally blocked by acetylation.
  • Arrays of overlapping 15-mer peptides, staggered at 5-residue intervals (Intavis, Germany) representing the entire length of the spike protein of SARS-CoV-2 were probed with serum pools (10 sera in each pool) from pre-COVID-19 or from patients convalescent (28 days post- symptomatic) who had tested positive by PCR for COVID-19 as described above.
  • serum pools (10 sera in each pool) from pre-COVID-19 or from patients convalescent (28 days post- symptomatic) who had tested positive by PCR for COVID-19 as described above.
  • a number of peptide epitopes were found that were recognized by multiple post-COVID pools that were not recognized by preimmune serum pools. In several cases these positive peptides represented neighbouring peptides in the array allowing the inference of a shared epitope sequence common to both, and mutual confirmation of the positive signal.
  • This peptide though not universally recognized by convalescent sera, is of significant diagnostic utility because it is highly specific for SARS-CoV-2, and not present in SARS-CoV-1 or in seasonal coronaviruses. Antibodies against this peptide may be further indicative of protection, since once bound, they would prevent spike activation by TMPRSS2, which is an activating protease that recognizes this site.
  • Table 8. indicates peptides that were recognized selectively by COVID-19 convalescent sera. Epitope sequences (inferred by peptide overlap) are indicated in bold text.
  • 15-mer peptides were made with various tags by Merrifield solid-state synthesis, incorporating (in some cases) an oligo-glycine (2mer) or PEG-6 spacer between the 15-mer and a C- terminal hexahistidine tag. These peptides were evaluated for their utility in ELISA tests and lateral flow devices in the examples that follow.
  • Figure 8 demonstrates specific reactivity of tagged peptides derived from microarray peptides E16 (SEQ ID NO: 6), E20 (SEQ ID NO: 8) and E19 (SEQ ID NO: 7) with COVID convalescent as distinct from control sera, in six pools, with ten sera per pool. Sera were tested at a dilution of 1/1000. All peptides have hexa-histidine tags but here are bound direct to unmodified polystyrene ELISA plates. All three peptides demonstrate specific recognition by convalescent sera (positive pool), over control serum pools.
  • E20 denotes N-Acetyl E20 with a C-terminally located hexa-histidine tag. The other peptides have a PEG-6 amino-acid in line with the other amino acids of the amino acid sequence, separating the viral peptide from the hexahistidine tag.
  • Tables 9 and 10 show lateral flow test results with serum pools, generated with peptide antigens derived from array peptides E16 (SEQ ID NO: 6), E20 (SEQ ID NO: 8) and G19 (SEQ ID NO: 13).
  • Tables 9 and 10 demonstrate the results of lateral flow tests conducted with select C- terminally His-tagged peptides probed with pooled pre-COVID and COVID-convalescent sera, using LF tests in the test architecture of Figure 5 using materials and reagents of Table 1 and Table 6 conditions “B”, essentially as described in Table 7, in which, instead of using RBD6His (SEQ ID NO:3) as antigen, the peptide antigens were the C-terminally-6His-tagged peptide antigens derived from array peptides E16 (SEQ ID NO: 6), E20 (SEQ ID NO: 8) and G19 (SEQ ID NO: 13), with a PEG6 spacer between the peptide and C-terminal-6His tag and wherein particle arrest (depending on the presence of human anti-peptide/anti-CoV-2 antibodies of the test sample) is achieved by agency of a murine monoclonal anti-His tag antibody on the test line.
  • RBD6His SEQ ID NO
  • G19 peptide with PEG-6 spacer and 6His derived from G19 peptide (SEQ ID NO: 13) (Acetyl-KPSKRSFIEDLLFNK-PEG-6-HHHHHH-COOH), very strong signals in convalescent sera, and excellent discrimination in signal between pre-COVID and COVID- convalescent sera were observed, despite the presence of a peptide representing only one epitope in the test. While in ELISA tests E20 and G19 performed similarly, in the LF tests G19 gave stronger signals.
  • E20 still gave excellent discrimination between SARS- CoV-2 PCR-positive (COVID-convalescent) and negative sera (pre-COVID) sera (especially when embodied with a PEG-6 spacer amino-acid between the viral peptide and the C- terminally located His6-tag.
  • pre-COVID negative sera
  • the E16-derived peptide did not discriminate in lateral flow tests between pre- and post-COVID sera each giving strong signals.
  • the ACE2 receptor-binding domain ‘RBD’ of SARS-CoV-2 spike protein (PMID 32225176) comprising residues R319-F541 (SEQ ID NO: 1) (of reference sequence YP_009724390.1 , which is ‘REFSEQ: accession NC_045512.2’ SEQ ID NO: 1
  • RV QPTESIVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN 361 CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD 421 YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC 481 NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV WLSFELLHA PATVCGPKKS TNLVKNKCVN 541 F
  • RBD-6His (numbering as per reference sequence YP_009724390.1) (ACE2 receptorbinding residues are shown in bold)

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Abstract

L'invention concerne une bandelette réactive à écoulement latéral pour la détection d'anticorps comprenant : (a) un tampon d'échantillon comprenant un ou plusieurs peptides antigéniques recombinants ou synthétiques et/ou une ou plusieurs protéines recombinantes comprenant une séquence d'acides aminés native de la cible, ledit peptide et/ou ladite protéine ayant une étiquette de liaison par affinité, dans lequel ladite cible n'est pas un Flavivirus, ladite cible étant choisie parmi un agent de maladie infectieuse et un marqueur auto-immun ; (b) un tampon conjugué comprenant un conjugué détecteur destiné à marquer les complexes immuns formés entre l'anticorps de la cible et l'antigène marqué de la cible ; (c) une bande de capture comprenant un moyen de capture destiné à capturer et immobiliser les complexes immuns marqués par le conjugué détecteur via le marqueur de liaison par affinité et (d) un tampon absorbant, les tampons et la bande étant disposés pour permettre une communication par écoulement capillaire entre eux.
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WO2024026553A1 (fr) * 2022-08-03 2024-02-08 Centre Hospitalier De L'université De Montréal Nouvel épitope antigénique anti-sars-cov-2 et ses utilisations

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