METHOD OF IDENTIFYING OR CHARACTERISING AN IMMUNE RESPONSE IN A SUBJECT

Abstract

This invention provides a method of identifying or characterising an immune response in a subject comprising: (a) contacting a sample containing immunoglobulins from the subject with at least one antigen immobilised on a support; (b) washing unbound, non-antigen specific immunoglobulins from the support to leave antigen-specific immunoglobulins bound to the antigen on the support; (c) optionally eluting the antigen-specific immunoglobulins from the antigen on the support; and (d) subjecting the antigen-specific immunoglobulins to mass spectrometry to identify two or more different antigen specific immunoglobulin classes, subclasses and/or light chain types.

Claims

1. A method of identifying or characterising an immune response in a subject comprising: (a) contacting a sample containing immunoglobulins from the subject with at least one antigen immobilised on a support; (b) washing unbound, non-antigen specific immunoglobulins from the support to leave antigen-specific immunoglobulins bound to the antigen on the support; (c) optionally eluting the antigen-specific immunoglobulins from the antigen on the support; and (d) subjecting the antigen-specific immunoglobulins to mass spectrometry to identify two or more different antigen specific immunoglobulin classes, subclasses and/or light chain types.

2. The method according to claim 1, wherein the sample is contacted with at least two different antigens, wherein each antigen on a different support or a different portion of the same support, and wherein the sample is optionally separated into at least two aliquots, and each aliquot is contacted with a different antigen bound to a different support.

3-4. (canceled)

5. The method according to claim 1, wherein (a) at least a portion of the antigen-specific immunoglobulins are subjected to a proteolytic digestion prior subjecting the digested antigen-specific immunoglobulins to the mass spectrometry; (b) at least a portion of the antigen-specific immunoglobulins are not subjected to a proteolytic digestions prior to subjecting the antigen-specific immunoglobulins to the mass spectrometry; or (c) at least a portion of the antigen-specific immunoglobulins are dissociated with at least one reducing agent and/or denaturing agent to separate light chains bound to heavy chains prior to subjecting the separated immunoglobulins to the mass spectrometry.

6-7. (canceled)

8. The method according to claim 1, wherein (a) the relative amount of the two or more of the different antigen specific immunoglobulin classes, subclasses and/or light chain types are compared to each other, or the amount of each of two or more of the different antigen specific immunoglobulin classes, subclasses and/or light chain types in the sample are determined; or (b) the amount of one or more different antigen specific immunoglobulin classes, subclasses and/or light chain types in the sample is quantitated.

9. (canceled)

10. The method according to claim 1, (a) wherein the immunoglobulin classes are selected from IgG, IgA, IgM, IgD and IgE; (b) wherein the immunoglobulin subclasses are selected from IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2; (c) wherein the immunoglobulin light chains are selected from lambda light chains and kappa light chains; (d) wherein the ratio of the relative amount of lambda : kappa light chains in the sample is determined; (e) wherein the method additionally comprises identifying one or more of a) J-chains bound to IgA and/or IgM and/or b) CD5L-bound to IgM; (f) wherein the immunoglobulins in the sample are purified or enriched, prior to contacting the antigen bound to the support; or (g) wherein at least a portion of the IgG in the sample is removed prior to contacting the remaining immunoglobulins with the antigen bound to the support.

11-16. (canceled)

17. The method according to claim 1, wherein, in step (c) antigen-specific immunoglobulins having lower antigen binding specificity are eluted from the antigen bound to the support prior to those having a higher antigen binding specificity.

18. The method according to claim 17, wherein, the or each antigen is an antigen from a virus, a bacterium, an archaebacterium, a fungus, a protozoan, a helminth, an autoimmune antigen, a cancer antigen or an antigen capable of inducing an allergic reaction in the subject.

19. The method according to claim 1, wherein at least a portion of the IgG in the sample is removed prior to contacting the remaining immunoglobulins with the antigen bound to the support and wherein the virus is a Coronavirus.

20. The method according to claim 17, wherein the virus antigen is at least an antigenic portion of a viral envelope protein, a capsid protein, an enzyme or haemagglutinin.

21. The method according to claim 17, wherein at least a portion of the IgG in the sample is removed prior to contacting the remaining immunoglobulins with the antigen bound to the support and wherein the bacterial antigen is at least one antigen portion of a cellular antigen, a flagella antigen, a somatic antigen, a virulence antigen, a fimbrial antigen or a toxoid.

22. The method according to claim 1, wherein the subject is a fish, a mammal, a bird, or a reptile, wherein the mammal is optionally selected from a human, a non-human ape, a monkey, a horse, a sheep, a camelid, a goat, a cow, a dog, a cat, or a rodent.

23. (canceled)

24. The method according to claim 17, (a) wherein the sample is a sample of biological fluid, typically blood, serum, plasma, cerebrospinal fluid, urine, tear, sputum, lavage fluid, or saliva; (b) wherein the support is selected from paramagnetic beads and a MALDI-TOF target; (c) wherein one or more additional indicators of the immune response in the subject is additionally determined; (d) wherein the mass spectrometry is Liquid Chromatography Mass Spectrometry or MALDI-TOF mass spectrometry; or (e) further comprising adding a predetermined amount of a calibrator to the sample.

25. (canceled)

26. The method according to claim 17, wherein an ionisation control is added to the sample, prior to carrying out the mass spectrometry.

27-28. (canceled)

29. The method according to claim 1, and further comprising producing a matrix to characterise an immune response in the subject by measuring of an amount of two or more two or more of the different antigen specific immunoglobulin classes, subclasses and/or light chain types compared to two or more different antigens.

30. (canceled)

31. The method according to claim 1, and further comprising (a) identifying an the-immune status of the subject; (b) characterising the immune response in the subject to a pathogen, allergen or other antigen; (c) selecting one or more vaccine targets, and determining severity or progression of a condition caused by a pathogen; (d) characterising an autoimmune response in the subject; (e) characterising an allergic response in the subject; (f) monitoring the immune response or progression of a disease in the subject; or (g) selecting a monoclonal antibody class or monoclonal antibody characteristics.

32-37. (canceled)

38. The method according to claim 1, whereby the neutralizing capacity of an immunoglobulin is determined by measuring molecules in the sample buffer indicative of neutralization of the bound antigen.

39. The method according to claim 1, to identify activating and non-activating antibodies for receptor sites.

40. A computer implemented method for identifying or characterising an immune response in a subject, comprising comparing a mass spectrum obtained for a first antigen specific immunoglobulin class, subclass and/or light chain type with a mass spectrum for a second antigen specific immunoglobulin class, subclass and/or light chain type, wherein the mass spectrum is obtained by a method according to claim 1.

41. The computer implemented method according to claim 40, wherein the computer comprises a computer processor and a computer memory.

42. An apparatus for identifying or characterising an immune response in a subject by a method according to claim 1, comprising the use of a computer implemented method comprising comparing a mass spectrum obtained for a first antigen specific immunoglobulin class, subclass and/or light chain type with a mass spectrum for a second antigen specific immunoglobulin class, subclass and/or light chain type, wherein the apparatus optionally includes a mass spectrometer.

43-44. (canceled)

Description

[0077] The invention will now be described by way of example only with reference to the following figures:

[0078] FIG. 1 shows five mass spectra for immunoglobulins which have been immunopurified with anti-IgG, anti-IgA, anti-IgM, anti-kappa and anti-lambda antibodies. After immunopurification, the heavy chains and light chains were dissociated using a reducing agent, dithiothreitol, prior to performing mass spectrometry using MALDI-TOF.

[0079] FIG. 2 is from Ladwig, P.M. etal., Clinical Chemistry (2014), Volume 16, pages 1080-1088. It shows that an LC-MS/MS ion chromatogram of an IgG sub-low control (The Binding Site Limited, Birmingham, United Kingdom) diluted in 1:16 bovine serum showing a peak (shaded) used with quantification of each individual subclass peptide, the common peptide used for IgG total, and the horse IgG peptide used for quantification. A number of non-specific background peaks seen were chromatographically separated and did not interfere.

[0080] FIG. 3 shows MALDI-TOF spectra using a Coronavirus viral spike protein immobilised in a paramagnetic bead. The spectra are presented over an extended 7000-30000 m/z range (showing all 3 charge states, A) and reduced 11000 to 14000 m/z range (swing only the +2-charge state, B). A monoclonal antibody (specific to the spike protein) was bound by the bead, eluted, and peak separated via MALDI-TOF mass spectrometry. Additionally, healthy human plasma and serum were also separately incubated with the viral spike protein bead. The bead marked viral spike protein (pre-cleared) was a control to demonstrate that post-conjugation bead washing did not damage the immobilised protein. The bead marked α-human IgG is a bead in which the viral protein has been replaced by an α-IgG-specific antibody to demonstrate that that antibody binds not only the monoclonal antibody, which is an IgG monoclonal antibody, but when used with normal plasma and normal serum, also binds to the immunoglobulins in the normal plasma and normal serum.

[0081] FIG. 4 MALDI mass spectrometry spectra from Covid-19 negative (healthy) and PCR-positive (diseased) individuals tested against viral SARS-CoV-2 Spike protein and bacterial Pneumococcal cell wall polysaccharide (CWPS) . Serum or plasma samples of 4 individuals were immunocaptured with antigen conjugated beads Individuals 2 and 4 were “diseased” (tested positive for Covid-19). Individuals 1 and 3 were classes “healthy” (tested negative for Covid-19).

[0082] FIG. 5 MALDI mass spectrometry spectra from healthy and diseased individuals tested against a targeted infection using various infection specific antigens. SARS-CoV-2 Virus specific antigens were conjugated to beads with the viral Spike protein and Nucleocapsid protein. Samples of 4 individuals were immunocaptured with antigen conjugated beads. Two of the individuals were “diseased” (tested positive for Covid-19, individual 2 and 4) and two healthy (individuals 1 and 3).

[0083] FIG. 6 Extracted ion chromatograms (XIC) of IgG1 peptide TPEVTC(CAM)VVVDVSHEDPEVK detected within the digest of ERM-DA470k reference serum and eluate digests of serum and plasma samples immunoprecipitated using beads conjugated with SARS-CoV-2 spike protein. (A) XIC of IgG1 peptide in ERM-DA470k. (B-E) XICs of IgG1 peptide in eluate digests of immunoprecipitated (B) COVID-19 negative serum, (C) COVID-19 negative plasma, (D) COVID-19 positive serum, and (E) COVID-19 positive plasma. *(CAM) = carbamidomethylated cysteine.

[0084] FIG. 7 Extracted ion chromatograms (XIC) of IgG2 peptide GLPAPIEK detected within the digest of ERM-DA470k reference serum and eluate digests of serum and plasma samples immunoprecipitated using beads conjugated with SARS-CoV-2 spike protein. (A) XIC of IgG2 peptide in ERM-DA470k. (B-E) XICs of IgG2 peptide in eluate digests of immunoprecipitated (B) COVID-19 negative serum, (C) COVID-19 negative plasma, (D) COVID-19 positive serum, and (E) COVID-19 positive plasma.

[0085] FIG. 8 Extracted ion chromatograms (XIC) of IgG3 peptide TPEVTC(CAM)VVVDVSHEDPEVQFK detected within the digest of ERM-DA470k reference serum and eluate digests of serum and plasma samples immunoprecipitated using beads conjugated with SARS-CoV-2 spike protein. (A) XIC of IgG3 peptide in ERM-DA470k. (B-E) XICs of IgG3 peptide in eluate digests of immunoprecipitated (B) COVID-19 negative serum, (C) COVID-19 negative plasma, (D) COVID-19 positive serum, and (E) COVID-19 positive plasma. *(CAM) = carbamidomethylated cysteine.

[0086] FIG. 9 Extracted ion chromatograms (XIC) of IgG4 peptide GLPSSIEK detected within the digest of ERM-DA470k reference serum and eluate digests of serum and plasma samples immunoprecipitated using beads conjugated with SARS-CoV-2 spike protein. (A) XIC of IgG4 peptide in ERM-DA470k. (B-E) XICs of IgG4 peptide in eluate digests of immunoprecipitated (B) COVID-19 negative serum, (C) COVID-19 negative plasma, (D) COVID-19 positive serum, and (E) COVID-19 positive plasma.

[0087] FIG. 10 Extracted ion chromatograms (XIC) of IgA1 peptide DASGVTFTWTPSSGK detected within the digest of ERM-DA470k reference serum and eluate digests of serum and plasma samples immunoprecipitated using beads conjugated with SARS-CoV-2 spike protein. (A) XIC of IgA1 peptide in ERM-DA470k. (B-E) XICs of IgA1 peptide in eluate digests of immunoprecipitated (B) COVID-19 negative serum, (C) COVID-19 negative plasma, (D) COVID-19 positive serum, and (E) COVID-19 positive plasma.

[0088] FIG. 11 Extracted ion chromatograms (XIC) of IgA2 peptide DASGATFTWTPSSGK detected within the digest of ERM-DA470k reference serum and eluate digests of serum and plasma samples immunoprecipitated using beads conjugated with SARS-CoV-2 spike protein. (A) XIC of IgA2 peptide in ERM-DA470k. (B-E) XICs of IgA2 peptide in eluate digests of immunoprecipitated (B) COVID-19 negative serum, (C) COVID-19 negative plasma, (D) COVID-19 positive serum, and (E) COVID-19 positive plasma.

[0089] FIG. 12 Extracted ion chromatograms (XIC) of kappa LC peptide SGTASVVC(CAM)LLNNFYPR detected within the digest of ERM-DA470k reference serum and eluate digests of serum and plasma samples immunoprecipitated using beads conjugated with SARS-CoV-2 spike protein. (A) XIC of kappa LC peptide in ERM-DA470k. (B-E) XICs of kappa LC peptide in eluate digests of immunoprecipitated (B) COVID-19 negative serum, (C) COVID-19 negative plasma, (D) COVID-19 positive serum, and (E) COVID-19 positive plasma. *(CAM) = carbamidomethylated cysteine.

[0090] FIG. 13 Extracted ion chromatograms (XIC) of lambda LC peptide YAASSYLSLTPEQWK detected within the digest of ERM-DA470k reference serum and eluate digests of serum and plasma samples immunoprecipitated using beads conjugated with SARS-CoV-2 spike protein. (A) XIC of lambda LC peptide in ERM-DA470k. (B-E) XICs of lambda LC peptide in eluate digests of immunoprecipitated (B) COVID-19 negative serum, (C) COVID-19 negative plasma, (D) COVID-19 positive serum, and (E) COVID-19 positive plasma.

[0091] FIG. 14 Extracted ion chromatograms (XIC) of IgG peptide DTLMISR detected within the digest of ERM-DA470k reference serum and eluate digests of serum and plasma samples immunoprecipitated using beads conjugated with SARS-CoV-2 spike protein. (A) XIC of IgG peptide in ERM-DA470k. (B-E) XICs of IgG peptide in eluate digests of immunoprecipitated (B) COVID-19 negative serum, (C) COVID-19 negative plasma, (D) COVID-19 positive serum, and (E) COVID-19 positive plasma.

[0092] FIG. 15 Extracted ion chromatograms (XIC) of IgA peptide SGNTFRPEVHLLPPPSEELALNELVTLTC(CAM)LAR detected within the digest of ERM-DA470k reference serum and eluate digests of serum and plasma samples immunoprecipitated using beads conjugated with SARS-CoV-2 spike protein. (A) XIC of IgA peptide in ERM-DA470k. (B-E) XICs of IgA peptide in eluate digests of immunoprecipitated (B) COVID-19 negative serum, (C) COVID-19 negative plasma, (D) COVID-19 positive serum, and (E) COVID-19 positive plasma. *(CAM) = carbamidomethylated cysteine.

[0093] FIG. 16 Extracted ion chromatograms (XIC) of IgM peptide GVALHRPDVYLLPPAR detected within the digest of ERM-DA470k reference serum and eluate digests of serum and plasma samples immunoprecipitated using beads conjugated with SARS-CoV-2 spike protein. (A) XIC of IgM peptide in ERM-DA470k. (BE) XICs of IgM peptide in eluate digests of immunoprecipitated (B) COVID-19 negative serum, (C) COVID-19 negative plasma, (D) COVID-19 positive serum, and (E) COVID-19 positive plasma.

[0094] FIG. 17 Fragmentation spectrum corresponding to IgG1 peptide TPEVTC(CAM)VVVDVSHEDPEVK within digested eluate of COVID-19 positive serum immunoprecipitated with beads conjugated with SARS-CoV-2 spike protein. (A) Fragmentation spectrum highlighting the C-terminal fragment ions (y-ion series). (B) Table of expected and observed y-ion series fragments. *(CAM)= carbamidomethylated cysteine.

[0095] FIG. 18 Fragmentation spectrum corresponding to IgG2 peptide GLPAPIEK within digested eluate of COVID-19 positive serum immunoprecipitated with beads conjugated with SARS-CoV-2 spike protein. (A) Fragmentation spectrum highlighting the C-terminal fragment ions (y-ion series). (B) Table of expected and observed y-ion series fragments.

[0096] FIG. 19 Fragmentation spectrum corresponding to IgG3 peptide TPEVTC(CAM)VVVDVSHEDPEVQFK within digested eluate of COVID-19 positive serum immunoprecipitated with beads conjugated with SARS-CoV-2 spike protein. (A) Fragmentation spectrum highlighting the C-terminal fragment ions (y-ion series). (B) Table of expected and observed y-ion series fragments. *(CAM)= carbamidomethylated cysteine.

[0097] FIG. 20 Fragmentation spectrum corresponding to IgG4 peptide GLPSSIEK within digested ERM-DA470k reference serum. (A) Fragmentation spectrum highlighting the C-terminal fragment ions (y-ion series). (B) Table of expected and observed y-ion series fragments.

[0098] FIG. 21 Fragmentation spectrum corresponding to IgA1 peptide DASGVTFTWTPSSGK within digested eluate of COVID-19 positive serum immunoprecipitated with beads conjugated with SARS-CoV-2 spike protein. ((A) Fragmentation spectrum highlighting the C-terminal fragment ions (y-ion series). (B) Table of expected and observed y-ion series fragments.

[0099] FIG. 22 Fragmentation spectrum corresponding to IgA2 peptide DASGATFTWTPSSGK within digested eluate of COVID-19 negative serum immunoprecipitated with beads conjugated with SARS-CoV-2 nucleocapsid protein. (A) Fragmentation spectrum highlighting the C-terminal fragment ions (y-ion series). (B) Table of expected and observed y-ion series fragments.

[0100] FIG. 23 Fragmentation spectrum corresponding to kappa LC peptide SGTASVVC(CAM)LLNNFYPR within digested eluate of COVID-19 positive serum immunoprecipitated with beads conjugated with SARS-CoV-2 spike protein. (A) Fragmentation spectrum highlighting the C-terminal fragment ions (y-ion series). (B) Table of expected and observed y-ion series fragments. *(CAM)= carbamidomethylated cysteine.

[0101] FIG. 24 Fragmentation spectrum corresponding to lambda LC peptide YAASSYLSLTPEQWK within digested eluate of COVID-19 positive serum immunoprecipitated with beads conjugated with SARS-CoV-2 spike protein (A) Fragmentation spectrum highlighting the C-terminal fragment ions (y-ion series). (B) Table of expected and observed y-ion series fragments.

[0102] FIG. 25 Fragmentation spectrum corresponding to IgG peptide DTLMISR within digested eluate of COVID-19 positive serum immunoprecipitated with beads conjugated with SARS-CoV-2 spike protein. (A) Fragmentation spectrum highlighting the C-terminal fragment ions (y-ion series). (B) Table of expected and observed y-ion series fragments.

[0103] FIG. 26 Fragmentation spectrum corresponding to IgA peptide SGNTFRPEVHLLPPPSEELALNELVTLTC(CAM)LAR within digested eluate of COVID-19 positive serum immunoprecipitated with beads conjugated with SARS-CoV-2 spike protein. (A) Fragmentation spectrum highlighting the C-terminal fragment ions (y-ion series). (B) Table of expected and observed y-ion series fragments. *(CAM) = carbamidomethylated cysteine.

[0104] FIG. 27 Fragmentation spectrum corresponding to IgM peptide GVALHRPDVYLLPPAR within digested eluate of COVID-19 positive serum immunoprecipitated with beads conjugated with SARS-CoV-2 spike protein. (A) Fragmentation spectrum highlighting the C-terminal fragment ions (y-ion series). (B) Table of expected and observed y-ion series fragments.

[0105] FIG. 28 Bar charts comparing peak areas of markers representing immunoglobulin isotypes IgG/IgA/IgM, IgG subclasses, IgA subclasses and light chains within digested eluates of negative and positive COVID-19 serum samples immunoprecipitated with beads conjugated with SARS-CoV-2 spike protein. Peak areas of (A) IgG subclasses, (B) IgA subclasses, (C) light chains, and (D) IgG/IgA/IgM immunoglobulins.

[0106] FIG. 29 Bar charts comparing peak areas of markers representing immunoglobulin isotypes IgG/IgA/IgM, IgG subclasses, IgA subclasses and light chains within digested eluates of negative and positive COVID-19 plasma samples immunoprecipitated with beads conjugated with SARS-CoV-2 spike protein. Peak areas of (A) IgG subclasses, (B) IgA subclasses, (C) light chains, and (D) IgG/IgA/IgM immunoglobulins.

[0107] The IgG classes and light chain types may be identified using techniques generally known in the art.

[0108] The spectrum obtained for different Ig classes and light chain types, using MALDI-TOF spectra are shown, for example, in FIG. 1. This utilises immunopurified immunoglobulins which have been dissociated with dithiothreitol as generally known in the art. This demonstrates that it is possible to identify the different heavy chain classes and light chain types. Method or performing such assays are shown in, for example WO2015/154052

[0109] FIG. 2 is an example from Ladwig et al, Supra, which demonstrates that it is also possible to determine subclasses of different immunoglobulins using mass spectrometry and quantifying them. Methods for carrying out that assay are described in that paper for example

[0110] Such techniques in the current invention are applied to antigen-specific immunoglobulins. A sample containing the immunoglobulin is contacted with an antigen attached to a substrate, such as a paramagnetic bead. The antigen-specific antibodies are then washed to remove non-specific binding, and eluted from the bead, prior to mass spectrometry.

[0111] FIGS. 3 (A and B) shows an example of a Coronavirus viral spike protein, attached to a paramagnetic bead. The upper panel shows that the activating and non-activating antibodies for receptor sites monoclonal antibody specifically binds to the viral spike protein bead, and is then eluted and detected via mass spectrometry as distinct peaks for immunoglobulin light chain and heavy chain. Normal serum and normal plasma immunoglobulins do not bind to the viral spike protein, so are washed off the bead, and are therefore not detected in the elution. The middle panel shows that pre-washing of the viral spike protein bead, does not substantially affect the immobilised protein, and still allows binding of the monoclonal antibody to it. The lower panel shows that if the viral spike protein is substituted for an a-IgG-specific antibody, then that monoclonal antibody binds to the bead. When such beads are incubated with normal plasma or normal serum, the IgG antibodies within the plasma or serum are detected, resulting in the overlapping broad lower peaks that are only observed in the lower panel.

[0112] Table 3 shows an example of a matrix of the typical amounts of different IgG subclasses and IgG that may be raised, in this example, against different viral proteins. Such a matrix may be expanded to include other classes, sub-types or light chains types. For example, IgE may be focussed on, for example, an IgE-related chronic disease such as allergic asthma or chronic urticaria might be being studies.

[0113] Such a matrix may also be converted into graphical format or other formats to allow comparison of the data between different immunoglobulin classes, subclasses, or light chain types, and different antigens. It may be automatically populated using a suitable computed implemented method.

[0114] To illustrate our approach we have analysed the immune response in four samples; 2 “diseased” individuals (PCR positive for Covid-19) and 2 “healthy” (tested negative for Covid-19) to 3 different infection related antigens; SARS CoV-2 Spike and Nucleocapsid protein and Pneumococcal cell wall polysaccharide (CWPS). Serum (sample 1 and 2) or plasma samples (3 and 4) of these 4 individuals were immunocaptured with these 3 antigen conjugated beads. Antibodies and antigen specific proteins captured by the beads are eluted, reduced and analysed by MALDI-TOF MS. In addition, antibodies and antigen specific proteins captured by the beads are eluted and analysed by LC-MS following tryptic digestion. Tryptic peptides for IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgG, IgA, IgM heavy chains and kappa and lambda light chains were used to “profile” the immune response. For LC-MS analysis the international protein reference material Da470k was also run as a serum control.

METHODS

Immunoprecipitation and MALDI-TOF-MS

[0115] Antibodies and antigen-specific proteins were captured by antigen conjugated paramagnetic beads and eluted under reducing conditions to disassociate light chains from the heavy chains. Briefly, 50-150 .Math.l of beads were washed three times with phosphate buffered saline, 0.1% Tween-20 (PBST). Diluted sample was added to the beads and incubated at room temperature with shaking for 30 mins. The beads were washed three times with PBST and then three more times with standard deionised water. 50 .Math.L of 0.1% formic acid (LC-MS) or 5% acetic acid including reducing agent (MALDI-TOF) was used to elute the beads by incubation at room temperature with shaking for 15 mins. The elution was subsequently sandwich-spotted with MALDI matrix (α-Cyano-4-hydroxycinnamic acid) onto a MALDI-TOF target plate and dried. Mass spectra were acquired in positive ion mode on a Bruker Microflex MALDI-TOF-MS covering the m/z range of 5000 to 80,000 which includes the doubly charged ([M + 2H]2+, m/z 10900-12300) ions of the analyte (human kappa or lambda light chains). Light chain observed in the 2+ charge state can be sectioned into 3 regions specific to each light chain; Lambda (11200-11560 m/z), Kappa (11570-11850 m/z) and Heavy Kappa (11900-12400 m/z).

LC-MS/MS and Digest Conditions

[0116] The eluate was transferred to a fresh microfuge tube and neutralised by the addition of 1 M triethylammonium bicarbonate (TEAB). The sample was then reduced with 200 mM tris(2-carboxyethyl)phosphine (TCEP), neutral pH for 30 mins at 60° C. at 1000 rpm before being cooled to room temperature. Alkylation was performed by the addition of 375 mM iodoacetamide and incubated for 30 mins at room temperature in the dark. Enzymatic digestion was carried out with 2.5 .Math.L of 1 .Math.g/.Math.L trypsin and incubation of the sample for 2 hours at 37° C. at 1000 rpm. The digestion reaction was terminated with 1 .Math.L of 100% formic acid. Sample volume was reduced using a vacuum concentrator at 60° C. on aqueous mode, prior to analysis by liquid-chromatography coupled electrospray ionisation mass spectrometry (LC-ESI-MS).

[0117] Samples were analysed on a Xevo G2-XS QToF mass spectrometer coupled to an ACQUITY I-Class UPLC system (Waters Ltd., Wilmslow, UK). 10 .Math.L of the digested sample was injected onto an ACQUITY UPLC Peptide BEH C18, 130 Å, 1.7 .Math.m, 2.1 × 150 mm column (Waters Ltd., Wilmslow, UK) maintained at 40° C. with a flow rate of 0.2 mL/min. A gradient from 0.1% (v/v) formic acid in water (A) to 0.1% formic acid (v/v) in acetonitrile (B) was employed. (gradient: 0-1 min, 1 % B; 1-60 min, 40% B; 60-70 min, 60% B; 70 min, 95% B; 70-80 min, 95% B; 80 min 1% B; 80-90 min 1% B). Capillary voltage was set to 1.5 kV with a 40 V cone voltage. Source temperature was set to 120° C. and desolvation temperature was set to 250° C. Cone gas glow was maintained at 50 L/h and desolvation gas flow at 600 L/h. MS.sup.E was acquired over 90 mins scanning between 100-2000 m/z. Scans alternated between a low collisional energy of 6 eV for 0.5 sec and a high collisional energy ramp from 25 eV to 45 eV for another 0.5 sec. LockSpray™ was enabled and Leucine Enkephalin was measured for 0.25 seconds every minute at 3 kV capillary voltage and 30 V cone voltage. The ions monitored for MS.sup.E relative quantitation are outlined in Table 5. The fragmentation spectrum of each individual immune-marker peptide are shown in FIGS. 17 to 27.

RESULTS

Maldi-tof

[0118] The overall antibody response against antigen conjugated beads was measured as peak intensity (a.u.) and characterised by peak distribution from a MALDI-MS data. Most individuals tested against the bacterial polysaccharide present a low level of natural antibody response (FIG. 4). This excludes Individual 3 (Covid-19 positive), who had a significantly high response to the bacterial antigen suggesting a recent bacterial infection. Overall, the light chain distribution observed against the bacterial antigen was predominantly polyclonal and oligoclonal with a bias towards kappa light chain usage, particularly heavy kappa. The same individuals were tested against the viral SARS-Cov-2 spike protein (FIG. 4). Covid-19 positive individuals (2 and 4) displayed a high antibody response against the spike protein, consisting of an underlying polyclonal light chain distribution (smooth peaks) with oligoclonal light chains (sharp peaks). The “healthy” Covid-19 negative individuals (2 and 4) present with a baseline antibody response. Subsequently, we compared the immune response from the SARS-Cov-2 nucleocapsid protein to the spike protein to characterise the response to antigens that differ in size, structure, function, and localisation (FIG. 5). As observed with the spike protein, the antibody response from Covid-19 positive individuals (2 and 4) also elicits a high response against the nucleocapsid protein. The nucleocapsid response largely consisted of lambda light chains with few oligoclonal peaks (FIG. 5). The relative response from “healthy” Covid-19 negative individuals (1 and 3) against nucleocapsid protein is close to baseline (FIG. 5). The κ:λ ratio of all 4 samples against the bacterial Pneumococcal Cell wall polysaccharide had a high κ:λ ratio suggesting a kappa light-chain bias (Table 4). The κ:λ ratio for response against the viral protein antigens on bead (SAR-Cov-2 Spike and Nucleocapsid protein) were variable with no evidence for a bias. The two Covid-19 positive individuals (2 and 4) show a response against Nucleocapsid protein, with κ:λ ratio of <1 suggesting the response is dominated by lambda light chain usage (Table 4).

[0119] In summary, the MALDI-TOF analysis has provided an overview of the antibody response to bacterial and viral antigens and indicated differences in quantity and quality of the immune response between these 4 individuals.

Lc- Ms/ms

[0120] An immune-marker tryptic- peptide that was specific (diagnostic) for the immunoglobulin being detected was chosen for each human immunoglobulin (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgG, IgA, IgM heavy chains and kappa and lambda light chains, (Table 5). The amino acid sequence and identity of these peptides was confirmed using the respective fragmentation ion spectrum as indicated in FIGS. 17-27. In each case the MS spectrum (A) is accompanied by the fragmentation ion table (B). These peptides were selected based on their length, relative abundance, and that they only occurred once in the polypeptide sequence of the protein.

[0121] These peptides were used to profile the immune response in four human samples; 2 “disease-state” individuals (PCR positive for Covid-19) and 2 “healthy” (tested negative for Covid-19) to 3 different microbial infection related antigens; SARS CoV-2 Spike and Nucleocapsid protein and Pneumococcal CWPS. In each case the extracted ion chromatogram was obtained to identify the presence of that peptide, and the area-under-the-peak was calculated and used for as a surrogate marker for the comparative measurement of the intact immunoglobulin from which it originated. For example the extracted ion chromatograms obtained for these 4 human clinical samples against the SARS-Cov-2 Spike protein is shown for IgG1 (FIG. 6), IgG2 (FIG. 7), IgG3 (FIG. 8), IgG4 (FIG. 9), IgA1 (FIG. 10), IgA2 (FIG. 11), and kappa (FIG. 12) and lambda light chains (FIG. 13). Additionally, peptide markers for total IgG (FIG. 14), total IgA (FIG. 15) and total IgM (FIG. 16) are also obtained. For comparison the international protein reference material ERM-DA470k was independently digested and run as a serum immunoglobulin control. The relative abundance, seen in FIGS. 6-16, can be compared and used to profile the antibody response between healthy and disease-state samples This is shown graphically for serum (FIG. 28) and plasma (FIG. 29) matrices. These have been sorted for easy comparison into IgG (FIGS. 28A and 29A) and IgA subclasses (FIGS. 28B and 29B), immunoglobulin light chains (FIGS. 28C and 29C) and total immunoglobulin IgG, IgA, and IgM (FIGS. 28D and 29D). The immune response of the Covid-positive samples is much greater than that of the Covid-negative samples which is dominated by an IgG response. In terms of subclass response both IgG1 and IgA1 are the dominant subclasses present. The light chain usage is approximately the same between kappa and lambda. This mirrors that observed using the MALDI-TOF platform (Table 4).

[0122] Equivalent analyses of the immunoglobulins immuno-captured by the Nucleocapsid protein and Pneumococcal CWPS beads was also produced. This data is combined with that of the Spike protein in Table 6 and expressed as a relative abundance profile. As expected the antibody immune response to the nucleocapsid protein antigen was lower in the Covid- negative (healthy) patients compared to the positive (disease-state) ones. In contrast to the two SARS-CoV-2 antigens, the immune response to Pneumococcal CWPS was more balanced between Covid positive and negative patients but was strongly biased to an IgG2 and kappa-light chain response. This also supported the results observed using MALDI-TOF-MS.

[0123] In summary, the tryptic-peptide LC-MS/MS analysis has provided a detailed overview of the antibody response to bacterial and viral antigens and allowed the antibody immune response between these 4 individuals to be profiled.

TABLE-US-00001 General IgG1 IgG2 IgG3 IgG4 Molecular mass (kD) 146 146 170 146 Amino acids in hinge region 15 12 62 12 Inter-heavy chain disulfide bonds 2 4 11 2 Mean adult serum level (g/l) 6.98 3.8 0.51 0.56 Relative abundance (%) 60 32 4 4 Half-life (days) 21 21 7/~21 21 Placental transfer + + + + ++ ++/++++ +++ Antibody response to: Proteins ++ +/- ++ ++ Polysaccharides + +++ +/- +/- Allergens + (-) (-) ++ Complement activation C1q binding ++ + +++ - Fc receptors FcyRI +++ ++++ ++ FcyRIIa.sub.H131 +++ ++ ++++ ++ FcyRIIa.sub.R131 +++ + ++++ ++ FcyRIIb/c + ++ + FcyRIIIa.sub.F158 ++ ++++ - FcyRIIIa.sub.V158 +++ + ++++ ++ FcyRIIIb +++ + + + + - FcRn (at pH <6.5) +++ +++ ++/+++ +++

TABLE-US-00002 Protein Peptide Transitions IgG1 GPSVFPLAPSSK y8-y10 IgG2 GLPAPIEK y4-y6 IgG3 WYVDGVEVHNAK y6, y8, y9 IgG4 TTPPVLDSDGSFFLYSR y8,y10,y12 IgA1 TPLTATLSK y5-y7 IgA2 DASGATFTWTPSSGK* y7-y10 IgA1-2 WLQGSQELPR y6-y8 IgM DGFFGNPR y4-y6 κ LC TVAAPSVFIFPPSDEQLK* y8,y9,y11 λ LC AGVETTTPSK y5-y7 IgD EPAAQAPVK y5-y7 IgE GSGFFVFSR* y5-y7

TABLE-US-00003 IgG1 (mg/L) IgG2 (mg/L) IgG3 (mg/L) IgG4 (mg/L) IgG (mg/L) IgA1 (mg/L) IgA2 (mg/L) IgA (mg/L) IgM (mg/L) IgD (mg/L) IgE (mg/L) Spike 1 10 1 100 0.1 111 Spike 2 11 1 100 0.1 112 membrane 30 5 300 0.1 335 Envelope 40 1 500 0 541 Nucleocapsid 0.1 0.1 500 0 500 Whole Virus 100 20 2000 <1 2120 Virus lysate 300 40 2000 <1 2340

TABLE-US-00004 Lambda (κ:λ) ratio of antibody response Antigen conjugated on bead Samples Sample Type Sample description* K:L Ratio Bacterial (Pneumococcal Cell wall polysaccharide) Individual 1 Serum Healthy 4.11 Individual 2 Serum Diseased 4.31 Individual 3 Plasma Healthy 5.85 Individual 4 Plasma Diseased 5.81 Viral (SARS-CoV-2 Spike protein) Individual 1 Serum Healthy N/D Individual 2 Serum Diseased 1.76 Individual 3 Plasma Healthy N/D Individual 4 Plasma Diseased 2.43 Viral (SARS-CoV-2 Nucleocapsid protein) Individual 1 Serum Healthy N/D Individual 2 Serum Diseased 0.67 Individual 3 Plasma Healthy N/D Individual 4 Plasma Diseased 0.56 * Sample description of “Healthy” refers to individuals tested PCR negative for Covid-19 and “Diseased” were individuals that test PCR positive.

Antigen-specific immunoglobulins were captured and eluted under reducing conditions to disassociate light chains from the heavy chains. Eluents were analysed by MALDI-Mass spectrometry, focusing on the distribution of light chains in the 2+ charge state (11000-12500 m/z). light chain observed in the 2+ charge state can be sectioned into 3 regions specific to each light chain; Lambda (11200-11500 m/z), Kappa (11600-11850 m/z) and Heavy Kappa (11900-12200 m/z). Area under the peak for lambda and total kappa lambda was used to calculate the κ:λ ratio for all antibody response using a open source mmass software.

TABLE-US-00005 List of markers used for immunoglobulin isotypes IgG, IgA and IgM, IgG subclasses, IgA subclasses, and light chains Marker Peptide Retention time (min) Precursor ion (m/z) IgG1 TPEVTC.sub.(CAM)VVVDVSHEDPEVK 30.10 713.69 IgG2 GLPAPIEK 21.14 412.75 IgG3 TPEVTC.sub.(CAM)VVVDVSHEDPEVQFK 33.98 805.39 IgG4 GLPSSIEK 18.52 415.74 IgA1 DASGVTFTWTPSSGK 30.12 770.87 IgA2 DASGATFTWTPSSGK 27.04 756.85 Kappa LC SGTASVVC.sub.(CAM)LLNNFYPR 43.02 899.45 Lambda LC YAASSYLSLTPEQWK 35.15 872.43 IgG DTLMISR 21.35 418.22 IgA SGNTFRPEVHLLPPPSEELALNELVTLTC.sub.(CAM)LAR 48.82 894.22 IgM GVALHRPDVYLLPPAR 28.93 444.26

Retention time (minutes) and Precursor ion (mass: charge [m/z]) listed for each marker peptide used for relative quantitation. Peak identified within a retention tolerance of ± 0.1 minute and precursor ion tolerance of ± 0.1 m/z.

TABLE-US-00006 Relative quantitation of immunoglobulins IgG/IgA/IgM, IgG subclasses, IgA subclasses and light chains within digested eluates of serum and plasma samples negative or positive for COVID-19 against beads conjugated with SARS-CoV-2 spike protein, SARS-CoV-2 nucleocapaid protein or pneumococcal cell wall polysaccharide (Streptococcus pneumoniae) ERM-DA470k SARS-CoV-2 spike protein SARS-CoV-2 nucleocapsid protein Pneumococcal cell wall polysaccharide Serum Plasma Serum Plasma Serum Plasma Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive IgG1 70.2% 82.3% 94.4% 77.3% 92.6% 77.9% 88.0% 82.3% 93.1% 17.8% 30.1% 9.9% 7.2% IgG2 22.9% 15.0% 2.4% 15.8% 1.0% 11.9% 7.6% 15.0% 1.5% 79.7% 59.3% 39.3% 91.7% IgG3 3.8% 2.7% 3.2% 6.5% 6.4% 7.3% 3.6% 2.7% 5.3% 1.7% 8.8% 1.3% 0.6% IgG4 3.1% 0.0% 0.0% 0.5% 0.0% 3.3% 0.7% 0.0% 0.1% 0.9% 1.6% 0.1% 0.4% IgA1 90.8% 83.8% 99.3% 98.2% 94.6% 88.9% 93.4% 83.8% 91.0% 87.1% 87.4% 86.8% 32.2% IgA2 9.2% 15.2% 0.7% 1.9% 5.2% 11.1% 6.6% 16.2% 9.0% 12.9% 12.6% 13.2% 17.8% Kappa LC 60.8% 38.0% 53.9% 49.4% 50.0% 57.6% 31.5% 45.5% 18.0% 73.4% 65.2% 79.4% 78.6% Lambda LC 39.2% 62.0% 46.1% 50.6% 50.0% 42.4% 68.5% 54.5% 82.0% 26.6% 34.8% 20.6% 21.4% K:L 1.55 0.61 1.17 0.98 1.00 1.55 0.46 0.83 0.22 2.76 1.87 3.65 3.67 IgG 72.1% 47.7% 77.0% 22.8% 69 .6% 11.0% 59.5% 1.1% 70.4% 2.6% 2.2% 50.2% 50.5% IgA 19.1% 8.1% 11.7% 52.8% 6.2% 21.0% 7.2% 1.3% 5.7% 0.1% 7.0% 4.4% 2.4% IgM 8.8% 44.2% 11.4% 14.3% 24.2% 68.0% 33.3% 97.6% 23.9% 97.2% 90.8% 45.4% 47.2%

Percentage compositions were calculated from the peak area of the monoisotopic peak of the marker peptides. Distribution of immunoglobulin components are subdivided into five categories: IgG subclasses, IgA subclasses, light chains (LC), light chain (kappa to lambda) ratio, and immunoglobulin isotypes: IgG, IgA and IgM. The most abundant component for each category is highlighted in grey.