WO2022109751A1

WO2022109751A1 - Point-of-care testing for sars-cov antibodies

Info

Publication number: WO2022109751A1
Application number: PCT/CA2021/051704
Authority: WO
Inventors: Shun-cheng LI; Sally ESMAIL; Courtney VOSS; Xuguang LIU
Original assignee: The University Of Western Ontario
Priority date: 2020-11-27
Filing date: 2021-11-29
Publication date: 2022-06-02

Abstract

A method for detecting the presence of SARS-CoV neutralizing immunoglobulins in a sample from a subject, comprising the steps of: (a) contacting the sample with particles covered with the SARS-CoV receptor binding domain (RBD) of the Spike (S) protein (S-RBD) under conditions that allow the particles covered with the S-RBD to agglutinate in the presence of immunoglobulins against the S-RBD and obtaining a first agglutination score, (b) mixing particles covered with the S-RBD with angiotensin converting enzyme 2 (ACE2) to form a mixture, and (c) adding the mixture to another aliquot of the sample taken from the subject under the same agglutination conditions as in step (a), and obtaining a second agglutination score, wherein a reduction in the first agglutination score of the sample in step (a) relative to the second agglutination score in step (c) is indicative of the presence of the SARS-CoV neutralizing immunoglobulins in the sample.

Description

TITLE OF THE INVENTION

Point-of-Care Testing for SARS-CoV antibodies

FIELD OF THE INVENTION

The present invention relates to methods for the detection of SARS-CoV antibodies, and materials related thereto.

BACKGROUND OF THE INVENTION

Development of rapid point-of-care COVID-19 diagnostics for use at the community level remains a top priority in the global response to the COVID-19 pandemic¹. While capacity for detecting SARS-CoV-2 based on nucleic acid amplification (NAAT) has grown immensely and enabled effective public health responses, serologic testing for virus specific antibodies has not gained the same widespread application, due to concerns over sensitivity, specificity, cost and turn-around time¹’². Although NAAT is the current gold standard for diagnosing acute infection, it is not effective in identifying individuals who have recovered from previous infection³. Given that approximately 40% of infected individuals remain asymptomatic¹’⁴’⁵ _, large-scale antibody testing could help better establish the true extent of the COVID-19 pandemic, identifying disease hotspots and high-risk populations to enable more effective isolation and contact tracing¹’³’⁶ . Moreover, antibody testing may identify individuals with a strong neutralizing antibody response who may be suitable donors for convalescent plasma/serum therapy for the treatment of those with severe symptoms⁷ and monitor efficacy of vaccines and duration of antibody responses to vaccines.

To date, a number of antibody tests have been approved for emergency use in the US and Europe. These tests detect the IgG, IgM or IgA antibody against the spike (including the receptor binding domain, RBD) or nucleocapsid (N) protein of the SARS- CoV-2 virus by enzyme-linked immunosorbent assay (ELISA)³. ELISA-based antibody tests, which can be qualitative or quantitative, require specialized instruments and are usually performed in a lab by a trained technician. The sensitivity and specificity of different ELISA kits vary widely^8-10. To enable point-of-care (POC) testing, several rapid diagnostic tests (RDTs) based on lateral flow have been developed. Although the RDTs reduced the time of the antibody test to 10-30 minutes from 2-5 hours (for ELISA), they generally suffer from decreased sensitivity and specificity compared to ELISA-based assays¹’^11-13. Accordingly, there is a need in the art for rapid, reliable means for accurately detecting SARS-CoV antibodies in general, and SARS-CoV-2 antibodies in particular, as provided in the present disclosure.

SUMMARY OF THE INVENTION

In one embodiment, there is provided a method of detecting presence of SARS- CoV neutralizing immunoglobulins in a sample taken from a subject, the sample having immunoglobulins, comprising: (a) contacting an aliquot of the sample taken from the subject with particles covered with the SARS-CoV receptor binding domain (RBD) of the Spike protein (S-RBD) under conditions that allow the particles covered with the S-RBD to agglutinate in the presence of immunoglobulins against the S-RBD (agglutination conditions) and obtaining a first agglutination score, (b) mixing particles covered with the S-RBD with angiotensin converting enzyme 2 (ACE2) to form a mixture, and (c) adding the mixture to another aliquot of the sample taken from the subject under the same agglutination conditions as in step (a), and obtaining a second agglutination score, wherein a reduction in the first agglutination score of the sample in Step (a) relative to the second agglutination score of the ACE2 mixture in Step (c) is indicative of the presence of the neutralizing immunoglobulins in the sample taken from subject.

In one embodiment, the S-RBD is the S-RBD of SARS-CoV-2.

In another embodiment, the method of further comprises comparing the first agglutination score and the second agglutination score with the agglutination scores of control samples having known amounts of neutralizing immunoglobulins, thereby providing a measurement of the neutralizing immunoglobulins in the sample based on said comparison. In another embodiment, the particles are red blood cells, latex particles, polystyrene microspheres, or microspheres or particles made of other non-latex and non-polystyrene polymers,

In another embodiment, the sample includes one or more of blood, serum, plasma, urine, cerebrospinal fluid and saliva and derivatives thereof.

Another embodiment relates to a method for detecting a humoral immune response to SARS-CoV in a subject, comprising the steps of: (a) contacting a sample having immunoglobulins taken from said subject with particles covered with a SARS-CoV antigen; and (b) detecting occurrence of agglutination of the particles covered with the SARS-CoV antigen, wherein occurrence of agglutination of the particles covered with the SARS-CoV antigen indicates a positive humoral immune response to SARS-CoV in the subject.

In one embodiment, the SARS-CoV antigen comprises a spike (S) protein of the SARS-CoV, a part of the S protein, a receptor binding domain (RBD) of the S protein, an epitope of the S protein, a nucleocapsid (N) protein of SARS-CoV-2, a part of the N protein, an RNA binding domain of the N protein (N-RBD), an epitope of the N protein, a matrix (M) protein (aka E1 membrane glycoprotein) of the SARS-CoV, a part of the M protein, an epitope of the M protein, or any combination thereof.

In another embodiment, the SARS-CoV antigen is a single peptide antigen of a protein, or more than one copy of the same single peptide antigen in tandem, or a combination of two or more different single peptide antigens from the same protein or from a different protein of the SARS-CoV that are either arranged in tandem in a single polypeptide or are mixed together.

In another embodiment, the single peptide antigen is an epitope of the S protein, an epitope of the N protein or an epitope of the M protein.

In another embodiment, the single peptide antigen includes no more than 25 amino acid residues, or no more than 20 amino acid residues, or no more than 15 amino acid residues, or no more than 10 amino acid residues. In another embodiment, the epitope of the S protein, the epitope of the N protein or the epitope of the M protein is selected from the epitopes of Table 3.

In another embodiment, the antigen is an epitope selected from the group consisting of SEQ ID NOs: 48, 82, 86 and 97. In another embodiment, the antigen is an epitope is selected from the group consisting of SEQ ID NOs: 116 and 139.

In another embodiment, the antigen is an epitope of a variant of concern (VOC epitope) of the SARS-CoV to detect the VOC specific immunoglobulins.

In another embodiment, the VOC epitope is selected from the group consisting of SEQ ID NOs: 150-220 and 221-227.

In another embodiment, the particles include particles covered with two or more different SARS-CoV antigens.

In another embodiment, the particles include a population of particles covered with the S protein or an epitope of the S protein, a population of particles covered with N protein or an epitope of the N protein, and a population of particles covered with both the S and N protein or multiple epitopes from the S and N proteins.

In another embodiment, the particles are red blood cells or latex particles.

In another embodiment, the sample includes one or more of blood, serum, plasma, urine, cerebrospinal fluid and saliva and derivatives thereof. In another embodiment, the SARS-CoV is SARS-CoV-2.

Another embodiment relates to a method of measuring the level of immunoglobulins against a SARS-CoV in a sample from a subject that contains immunoglobulins, comprising the steps of: (a) contacting the sample with particles covered with a SARS-CoV antigen under conditions that allow the particles covered with the SARS-CoV antigen to agglutinate in the presence of immunoglobulins against SARS-CoV; and (b) detecting occurrence or absence of particle agglutination in the sample, said occurrence or absence of particle agglutination in the sample having an agglutination area, and correlating the agglutination area of the sample with the agglutination areas of multiple control agglutinations, each control agglutination area containing known concentrations of immunoglobulins against SARS-CoV, to provide a measure of the level of the immunoglobulins against the SARS-CoV in the sample.

In one embodiment, the SARS-CoV antigen comprises a spike (S) protein of SARS-CoV, a part of the S protein, a receptor binding domain (RBD) of the S protein, an epitope of the S protein, a nucleocapsid (N) protein of SARS-CoV, a part of the N protein, an RNA binding domain of the N protein (N-RBD), an epitope of the N protein, a matrix (M) protein (aka E1 membrane glycoprotein) of SARS-CoV, a part of the M protein, an epitope of the M protein, or any combination thereof.

In another embodiment, the SARS-CoV antigen is a single peptide antigen of a protein, or more than one copy of the same single peptide antigen in tandem, or a combination of different single peptide antigens from the same protein orfrom different proteins of SARS-CoV that are either arranged in tandem in a single polypeptide or are mixed together.

In another embodiment, the single peptide antigen includes no more than 25 amino acid residues, or no more than 20 amino acid residues, or no more than 15 amino acid residues, or no more than 10 amino acid residues.

In another embodiment, the epitope of the S protein, the epitope of the N protein or the epitope of the M protein is selected from the epitopes of Table 3.

In another embodiment, the particles include particles covered with different SARS- CoV antigens.

In another embodiment, the particles include a population of particles covered with the S protein, a part of the S protein, or an epitope of the S protein, a population of particles covered with the N protein, a part of the N protein or an epitope of the N protein, and a population of particles covered with both the S and N protein or multiple epitopes from the S and N proteins. In another embodiment, the antigen is an epitope selected from the group consisting of SEQ ID NOs: 82 and 97.

In another embodiment, the antigen is an epitope is selected from the group consisting of SEQ ID NOs: 116 and 139.

In another embodiment, the antigen is an epitope of a variant of concern (VOC epitope) of the SARS-CoV and the measure provides the level of VOC specific immunoglobulins against the SARS-CoV in the sample.

In another embodiment, the particles are red blood cells, latex particles, polystyrene microspheres, or microspheres or particles made of other non-latex and non-polystyrene polymers.

In another embodiment, each control agglutination containing the known concentration of the immunoglobulins against SARS-CoV is assigned a score between 0 and 4 based on the agglutination intensity, 0 corresponds to no agglutination, 1 corresponds to about 25% agglutination, 2 corresponds to about 50% agglutination, 3 corresponds to about 75% agglutination, and 4 corresponds to about 100% agglutination, and wherein step (c) further comprises assigning a score to the sample based on the comparison to the agglutination intensity of each control agglutination.

36. The method of any one of claims 21 to 35, wherein the SARS-CoV is SARS-

CoV-2. Another embodiment relates to a method for semi-quantitatively measure of a titer of antibody against a SARS-CoV in a subject, comprising the steps of: (a) contacting a sample containing immunoglobulins from said subject with particles covered with a SARS-CoV antigen; (b) allowing the particles covered with the SARS-CoV-2 antigen to agglutinate into a clump area; (c) calculating the percentage of agglutination based on agglutination/clumps area relative to the total particle reaction area, and (d) plotting the percentage of agglutination against an antibody titer curve to obtain the titer of antibody against the SARS-CoV in the subject.

In one embodiment, the SARS-CoV antigen comprises a spike (S) protein of the SARS-CoV, a part of the S protein, a receptor binding domain (RBD) of the S protein, an epitope of the S protein, a nucleocapsid (N) protein of SARS-CoV, a part of the N protein, an RNA binding domain of the N protein (N-RBD), an epitope of the N protein, a matrix (M) protein of the SARS-CoV, a part of the M protein, an epitope of the M protein (aka E1 membrane glycoprotein), or any combination thereof.

In another embodiment, the SARS-CoV antigen is a single peptide antigen of a protein, or more than one copy of the same single peptide antigen in tandem, or a combination of different single peptide antigens from the same protein orfrom different proteins of the SARS-CoV that are either arranged in tandem in a single polypeptide or are mixed together.

In another embodiment, the particles include particles covered with different SARS- CoV antigens. In another embodiment, the particles include a population of particles covered with the S protein, a part of the S protein or an epitope of the S protein, and a population of particles covered with the N protein, a part of the N protein or an epitope of the N protein, and a population of particles covered with both the S and N protein or multiple epitopes from the S and N proteins.

In another embodiment, the antigen is an epitope selected from the group consisting of SEQ ID NOs: 82 and 97.

In another embodiment, the antigen is an epitope is selected from the group consisting of SEQ ID NOs: 116 and 139. In another embodiment, the antigen is an epitope of a variant of concern (VOC epitope) of the SARS-CoV.

In another embodiment, the particles are red blood cells, latex particles, polystyrene microspheres, or particles made of other non-latex polymers and polystyrene polymers.

In another embodiment, the SARS-CoV is SARS-CoV-2. Another embodiment relates to a method for determining coronavirus disease 2019 (COVID-19) severity comprising: (a) collecting a sample from a subject whose COVID- 19 severity needs to be determined, (b) mixing the sample with particles coated with a SARS-CoV-2 epitope that binds to a SARS-CoV-2 immunoglobulin under conditions that promote agglutination of the particles coated with the SARS-CoV-2 epitope with SARS-CoV-2 immunoglobulin present in the sample (agglutination conditions), and measuring a degree of agglutination, (c) comparing the degree of agglutination obtained in step (b) with (i) a negative control degree of agglutination obtained by mixing a sample from an individual that is SARS-CoV-2 negative with the particles coated with a SARS-CoV-2 epitope that binds to a SARS-CoV-2 immunoglobulin under the same agglutination conditions, (ii) a moderate control degree of agglutination obtained by mixing a sample from an individual with known moderate COVID-19 symptoms with the particles coated with a SARS-CoV-2 epitope that binds to a SARS-CoV-2 immunoglobulin under the same agglutination conditions, and (iii) a severe control degree of agglutination obtained by mixing a sample from an individual with known severe COVID-19 symptoms with the particles coated with a SARS-CoV- 2 epitope that binds to a SARS-CoV-2 immunoglobulin under the same agglutination conditions, and (d) determining COVID-19 severity based on the comparison of step (c).

Another embodiment relates to a method for determining coronavirus disease 2019 (COVID-19) outcome comprising: (a) collecting a sample from a subject whose COVID-19 outcome needs to be determined, (b) mixing the sample with particles coated with a SARS-CoV-2 epitope that binds to a SARS-CoV-2 immunoglobulin under conditions that promote agglutination of the particles coated with the SARS- CoV-2 epitope with SARS-CoV-2 immunoglobulin present in the sample (agglutination conditions), and measuring a degree of agglutination, (c) comparing the degree of agglutination obtained in step (b) with (i) a negative control degree of agglutination obtained by mixing a sample taken from an individual that is SARS-CoV-2 negative with the particles coated with a SARS-CoV-2 epitope that binds to a SARS-CoV-2 immunoglobulin under the same agglutination conditions, (ii) a live control degree of agglutination obtained by mixing a sample taken from an individual with that is COVID- 19 positive with the particles coated with a SARS-CoV-2 epitope that binds to a SARS- CoV-2 immunoglobulin under the same agglutination conditions, and (iii) a fatal control degree of agglutination obtained by mixing a sample taken from an individual that died of COVID-19 with the particles coated with a SARS-CoV-2 epitope that binds to a SARS-CoV-2 immunoglobulin under the same agglutination conditions, and (d) determining COVID-19 outcome based on the comparison of step (c).

In one embodiment of the methods determining coronavirus disease 2019 (COVID- 19) severity and outcome, the SARS-CoV-2 epitope is one SARS-CoV-2 epitope or combination of two or more SARS-CoV-2 epitopes selected from the SARS-CoV-2 epitopes of Table 3.

In one embodiment of the methods determining coronavirus disease 2019 (COVID- 19) severity and outcome, the one SARS-CoV-2 epitope or combination of two or more

SARS-CoV-2 epitopes is selected from the group consisting of SEQ ID NOs: 48, 82, 86 and 116.

In one embodiment of the methods determining coronavirus disease 2019 (COVID- 19) severity and outcome, the antigen is an epitope of a variant of concern (VOC epitope) of the SARS-CoV and the measure provides the level of VOC specific immunoglobulins against the SARS-CoV in the sample.

In one embodiment of the methods determining coronavirus disease 2019 (COVID- 19) severity and outcome, the VOC epitope is selected from the group consisting of SEQ ID NOs: 150-220 and 221-227. Another embodiment relates to a method of identifying subjects that have been infected with SARS-CoV-2 and subjects that have been vaccinated with a COVID-19 vaccine and have not been infected with SARS-CoV-2, the method comprising: (a) mixing a sample taken from a subject with particles coated with SARS-CoV-2 nucleocapsid (N) protein under conditions that promote agglutination of the particles coated with the SARS-CoV-2 N protein with anti-N SARS-CoV-2 immunoglobulins, (b) mixing a sample taken from the same subject with particles coated with SARS-CoV-2 spike (S) protein under conditions that promote agglutination of the particles coated with the SARS-CoV-2 S protein with anti-S SARS-CoV-2 immunoglobulins, and (c) measuring a level of agglutination of the particles coated with SARS-CoV-2 N protein in (a) and a level of agglutination of the particles coated with SARS-CoV-2 S protein in (b), wherein when the level of agglutination of the particles coated with SARS-CoV- 2 N protein are detectable, then the subject has been infected with SARS-CoV-2, and when the level of agglutination of the particles coated with SARS-CoV-2 S protein are detectable, and the level of agglutination of the particles coated with SARS-CoV-2 N protein are not detectable, then the subject has been vaccinated but not infected with SARS-CoV-2.

In one embodiment, the S protein is a part of the S protein, a receptor binding domain (RBD) of the S protein or an epitope peptide of the S protein and the N protein is a part of the N protein, a RNA binding domain of the N protein or an epitope peptide of the N protein.

Another embodiment, relates to a system for detecting immunoglobulins against a target antigen (anti-target antigen immunoglobulins), the system comprising: (a) syringe having a tube that is open at a first end, and a plunger that is slidably mounted inside the tube through a second end of the tube, (b) dyed particles coated with target antigen (“non-agglutinated particles”) such that when the dyed particles are mixed with anti-target antigen immunoglobulins, the dyed particles coated with the target antigen and the anti-target antigen immunoglobulins form agglutinated particles within the tube, and (c) a polymer gel preloaded in the tube, the polymer gel having a pore size that allows separation of the agglutinated particles from the non-agglutinated particles when the plunger pushes the agglutinated particles towards the first end.

In one embodiment of the system, the tube is graded with marks that provide a semi-quantitative measurement of the detected immunoglobulins against the target antigen. In another embodiment of the system, the marks are graded using samples that produce a specific level of aggregation of about 25%, about 50%, about 75% and about 100%.

Another embodiment relates to a method to detect immunoglobulins against a target antigen (“anti-target antigen immunoglobulins”) in a sample that contains immunoglobulins taken from a subject comprising: (a) mixing the sample with dyed particles coated with the target antigen (“non-agglutinated particles”) to form a mixture, such that when the sample contains anti-target antigen immunoglobulins, the dyed particles coated with the target antigen and the anti-target antigen immunoglobulins form agglutinated particles, (b) loading the mixture to one end of a tube pre-loaded with a neutral hydrogel having a pore size for separating the dyed beads coated with the target antigen when agglutinated from dyed beads coated with the target antigen when non-agglutinated, and (c) pushing the mixture at the one end of the tube loaded with the neutral hydrogel towards a second end of the tube, wherein the agglutinated particles form a band of agglutinated particles, thereby detecting the immunoglobulins against the target antigen.

In one embodiment, the target antigen is an antigen from a pathogen of interest.

In another embodiment, the target antigen is a SARS-CoV antigen selected from a spike (S) protein of the SARS-CoV, a part of the S protein, a receptor binding domain (RBD) of the S protein, an epitope of the S protein, a nucleocapsid (N) protein of SARS-CoV-2, a part of the N protein, an RNA binding domain of the N protein (N- RBD), an epitope of the N protein, a matrix (M) protein (aka E1 membrane glycoprotein) of the SARS-CoV, a part of the M protein, an epitope of the M protein, or any combination thereof.

In another embodiment, the epitope of the S protein, the epitope of the N protein or the epitope of the M protein is selected from the epitopes of Table 3. In another embodiment, the tube is graded with marks that provide a semi- quantitative measurement of the immunoglobulins against the target antigen, and wherein the method further comprises correlating a position of the band within the tube with the marks to provide said semi-quantitative measurement of the immunoglobulins against the target antigen in the sample.

In another embodiment, a position of the band of agglutinated particles within the tube provides semi-quantitative measurement of the level of anti-target antigen immunoglobulins in the subject.

Another embodiment relates to an isolated SARS-CoV-2 epitope that binds to a SARS-CoV-2 antibody, wherein said SARS-CoV-2 epitope is selected from the group of epitopes listed in Table 3.

Another embodiment relates to an isolated SARS-CoV-2 epitope that binds to a SARS-CoV-2 antibody, wherein said SARS-CoV-2 epitope is selected from the group consisting of SEQ ID NOs: 150-220 and 221-227.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures illustrate various aspects and preferred and alternative embodiments of the invention.

Figs. 1A-1B. Illustration of the principle of agglutination assay for SARS-CoV-2 antibody testing. (A) Latex particles or red blood cells (RBCs) are surface-coated with a SARS-CoV-2 antigen, the S-RBD or nucleocapsid (N). Incubation with plasma or serum containing antibodies against the coated antigen would induce agglutination of the latex particles or RBCs. (B) A representative image of the agglutination assay using latex beads coated with S-RBD.

Figs. 2A-2D. Antibody-induced latex particle agglutination correlates with the antibody titer. (A, B) Changes in agglutination in response to increased concentrations of the anti-S-RBD (n=3) or anti-N antibody (n=3). Dash lines represent fitted curves to Hill equation (h, Hill coefficient). (C, D) S-RBD (C) or N (D) antibody- induced agglutination decreased with increased dilution of plasma. Shown are agglutination data from three COVID-19⁺ plasma samples with 1 :2 to 1 :128 dilution (in log2 scale; three replicates/concentration). A COVID-19 negative (COVID-19^") sample and a sample collected in 2018 (Pre-COVID-19) were included as controls.

Figs. 3A-3B. Agglutination assay distinguished COVID-19+ from COVID-19- samples. Comparison of S-RBD (A) and N (B) antibody responses between COVID- 19⁺ (n=169), COVID-19- (n=121) and Pre-COVID-19 (n=100) plasma or serum samples determined by the agglutination assay. Statistical analyses were performed using unpaired Student’s t-test with Welch's correction (p values shown on graph).

Fig. 4A-4B. Semi-quantitative measurement of anti-S and anti-N antibodies by agglutination scores. (A) Latex beads coated with the N or S-RBD antigen were mixed with the indicated concentration of anti-N (nucleocapsid) or anti-S-RBD IgG. Anti-S-RBD (monoclonal, NBP2-90980) was obtained from Novus Biologicals; Anti- Nucleocapsid (polyclonal, PA5-81794) was from ThermoFisher Scientific. Images were taken after the mixtures were incubated for 2 minutes and the area of agglutination quantified. The agglutination scores were assigned as 4 = 75-100% agglutination; 3 = 50-75% agglutination; 2 = 25-50% agglutination; 1 = 5-25% agglutination; 0 (or negative) <5% agglutination. Anti-S-RBD (monoclonal, NBP2- 90980) was obtained from Novus Biologicals; Anti-Nucleocapsid (polyclonal, PAS-

81794) was from ThermoFisher Scientific. (B) Representative images of agglutination of five samples with distinct agglutination scores (0-4) in the nucleocapsid (N) (upper row) or the S-RBD antibody (S-RBD) test (lower row).

Fig. 5A-5B. Semi-quantitative agglutination assay for SARS-CoV-2 antibodies. The strength of the S-RBD and N antibody response in the COVID-19⁺ (n=169) plasma samples were determined semi-quantitatively by the latex bead agglutination assay with scores (1-4 denotes weak-to-strong antibody response). The samples formed 4 distinct groups with different agglutination scores. P values shown were based on unpaired Student’s t-test with Welch's correction. Figs. 6A-6B. SARS-CoV2 antibody testing based on RBC agglutination. Red blood cells (RBC, group O; R2R2) carrying the D antigen were labeled with anti-D IgG conjugated to recombinant S-RBD or N-RBD through streptavidin-biotin (i.e. , IgG- streptavidin conjugated to biotin-RBD). (A) Spike RBD labeled RBCs were mixed with either SARS-CoV-2- (left) or SARS-CoV2⁺ plasma (right). (B) Nucleocapsid RBD labeled RBCs were mixed with either SARS-CoV2- (left) or SARS-CoV2⁺ plasma (right). Images shown were taken after 2 min incubation at room temperature.

Fig. 7. Antibody testing based on the agglutination of polybeads carboxylate red dyed microspheres coated with the S-RBD antigen. The purified S-RBD was immobilized on the beads covalently and mixed with plasma samples from SARS- CoV-2 negative or positive (based on PCR testing) individuals. Images shown were photographed after 2 minutes of incubation.

Fig. 8. Antibody testing by agglutination differentiate between COVID-19⁺ and vaccinated individuals. The graph shows the distribution of antibodies specific for the Spike Ectodomain (blue dots) or the nucleocapsid (N, red triangle) in the COVID- 19⁺ (n=5) and COVID-19- vaccinated (by the Pfizer-BioNTech mRNA vaccine BNT162b2; n=5) plasma samples determined by the Polybeads microsphere agglutination assay. Statistical analyses were performed using paired Student’s t-test with Welch's correction (p values shown on graph). Figs. 9A-9C. SDS-PAGE images of recombinant SARS-CoV-2 proteins employed in the current disclosure. (A) Nucleocapsid protein (residues 1-419) and Spike RBD (residues 319-541). (B) ACE2 (residues 1-615). (C) His6-TEV-N-RBD and N-RBD.

Figs. 10A-10C. A pseudo-neutralization antibody (Nab) assay based on ACE2- RBD competition ELISA and correlation of Nab with agglutination using the S- RBD antigen. (A) A schematic to illustrate the principle of the ELISA-based neutralization antibody test. The S-RBD (triangle shapes) is immobilized on an ELISA plate and the plate is then incubated with biotinylated ACE2. The ACE2-RBD binding is detected by streptavidin conjugated with horse radish peroxidase (HRP). The HRP signal will be reduced or blocked if neutralizing antibody (Nab) is present in the plasma (right). (B) Neutralization antibody response correlated significantly with the agglutination score determined using the S-RBD-beads. P values calculated based on unpaired Student t-test (n=10 for each group). (C) Spearman (r) correlation of efficiency of neutralization and S-RBD- or N-specific antibody response. Fig. 11. A schematic diagram to illustrate the principle of neutralizing antibody test based on agglutination of S-RBD-coated beads in the absence or presence of ACE2. In the absence of recombinant ACE2 (angiotensin converting enzyme 2), the agglutination of the S-RBD-coated beads measures the total amount of antibody present in the fluid sample, in this case peripheral blood (upper panel). In contrast, when ACE2 is preincubated with the S-RBD-beads, the RBD epitopes will be masked by the bound ACE2. This results in the reduction of agglutination induced by the neutralization antibodies (denoted by the Y symbol with arrow), but not by the non- neutralizing antibodies (denoted by the Y symbol).

Fig. 12A-12C. Representative data showing how agglutination assay in the presence of ACE2 can be used to measure the neutralizing antibodies. (A) Two

SARS-CoV-2-positive plasma samples with different percentages of neutralizing antibody (Nab) (-90% Nab for patient #1 and -10% for patient #2; determined by the ELISA-based assay, see also Fig. 10) were subjected to the agglutination assay using S-RBD-coated beads in the absence or presence of ACE2. The Nab (%) is determined by the equation: Nab (%) = %Aggregation (-ACE2) - %Aggregation (+ACE2). (B) Comparison of agglutination (%) with or without ACE2 for a group of 9 SARS-CoV-2- positive plasma samples. The p value shown is based on a paired t-test. (C) Correlation (simple line regression) between Nab values (percentage of total antibody) measured by the agglutination assay (+/-ACE2) or the ACE2-RBD competition ELISA assay (Fig. 10) for the same set of samples as in (B).

Fig. 13. Gel card agglutination assay to determine SARS-CoV-2 antibodies in a semi-quantitative manner. SARS-CoV-2 positive or negative (control, Ctrl) plasma samples were mixed with red-dyed polybeads (immobilized with S-RBD) for about 2 minutes before the mixture was transferred to the Neutral Gel card (Ortho-Clinical Diagnostics, Cat#MTS085014Ref#210343). Centrifugation of the gel card microtubes at 500 G force for 10 min resulted in the separation of agglutinated beads (in the SARS-CoV-2+ samples) from the non-agglutinated beads (in the negative control sample). The position of the beads in the column gel corresponds to the degree of agglutination, hence antibody titer (4+ being the strongest and 1+ the weakest).

Fig. 14A-14B. A syringe gel agglutination assay to determine SARS-CoV-2 antibodies in a semi-quantitative manner. (A) A schematic diagram to illustrate the principle and major steps of the syringe gel agglutination assay invention. In Step 1 , the sample (e.g., finger prick blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears and so forth) is diluted in a buffer or passed through plasma separation membrane (which lyse/separate the red blood cells in the case when whole blood is used in the assay) and mixed with or contacted with antigen (including epitope)-coated dyed beads (in this particular case the beads are red-dyed). The mixture is allowed to stand in room temperature (for example for 2 minutes or 5 minutes). In Step 2, the mixture is transferred to a syringe filled with hydrogels, matrigels, neutral gels or any gel or polymer with pore size suitable for separating the agglutinated from the non- agglutinated beads (e.g., 1-100 μm in diameter). The mixture may be added from the top or side or the syringe or drawn from the bottom of the syringe. In Step 3, the plunger of the syringe is pushed (if the mixture is added to the top of the gel/polymer) or pulled (if the mixture is drawn from the bottom of the gel/polymer) for a certain distance (determined by the length of the gel column and the sample volume). This pushing or pulling (as the case may be) of the plunger will result in the separation of the agglutinated beads (which cannot go through the gel/polymer) from the non- agglutinated beads (which are capable of penetrating the gel/polymer). The position of the band of beads provides a semi-quantitative measurement of antibody titer similar to that in the gel-card assay (see Fig. 12). (B) Photographs showing an example on how the syringe gel assay worked for a control sample (left) without anti- S antibody and a SARS-CoV-2+ sample with strong (4+) anti-S (spike) antibody response. Fig. 15A-15B. SARS-CoV2 antibody testing based on agglutination of latex particles conjugated with linear epitope peptide antigens. A linear epitope means a peptide with continuous sequence (usually 5-25 amino acids in length) in a protein antigen that the antibody binds to. (A) Streptavidin-conjugated latex particles were labeled with two biotinylated S antigen epitope peptides (2S; in 1 :1 molar ratio), two biotinylated N antigen epitope peptides (2N; 1 :1 ratio) or a mixture of the 2 S and 2 N antigen peptides in equal molar ratio (2N:2S). The epitope peptide conjugated latex beads were then mixed with either SARS-CoV2- orSARS-CoV2+ (based on PCR test) plasma. Images shown were taken after 5 min incubation at room temperature. Sequences of the two S epitope peptides used in this latex agglutination assay are: S- 811 : KPSKRSFIEDLLFNK and S-1146: DSFKEELDKYFKNHT. Sequences of the two N epitope peptides used in this latex agglutination assay are: N-156: AIVLQLPQGTTLPKG. N-361 : KTFPPTEPKKDKKKK. Combination of these two S and two N peptides (sequence listed above) were mixed as 1 :1 ratio. (B) Epitope- based latex agglutination assay distinguished COVID-19+ (n=20), COVID-19- (n=20) and Pre-COVID-19 (n=20). Statistical analyses were performed using unpaired Student’s t-test with Welch's correction (p values shown on graph). The epitope peptides used were: S-811 and S-1146 from the spike and N-156 and N-361 from the nucleocapsid protein. Figs. 16A-16G. Epitope-specific agglutination assay distinguished COVID-19 disease severity and outcome. (A-C) Correlation of disease severity with antibody responses to the S-811 , N-156 or N-361 epitope determined by latex bead agglutination. (D-G) Correlation of disease outcome with antibody responses to the S- 551 , S-811 , S-881 or N-156 epitope determined by latex bead agglutination. P values calculated based on unpaired one-tail Student t-test with Welch’s correction (no assumption of equal SD). *p<0.05, **p<0.002.

DESCRIPTION OF THE INVENTION

Definitions In this specification and in the claims that follow, reference will be made to several terms that shall be defined to have the meanings below. All numerical designations, e.g., dimensions and weight, including ranges, are approximations that typically may be varied ( + ) or ( - ) by increments of 0.1 , 1 .0, or 10.0, as appropriate. All numerical designations may be understood as preceded by the term “about”.

The term “about,” particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

The term “animal” includes humans and other animals.

The term “sample” includes a biological sample, including body fluid. The term "body fluid", as used herein, includes blood, serum, plasma, urine, cerebrospinal fluid, saliva and any other body fluid that includes immunoglobulins, and including derivatives of blood, serum, plasma, urine, cerebrospinal fluid or derivatives of any other body fluid that includes immunoglobulins.

The term “rapid” is meant to encompass a method that is completed (i.e. , a result is given) in about 5 minutes or less (i.e., 4 minutes, 3 minutes, 2, minutes, 1 minute and under 1 minute).

Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the terms “include”, “has” and their grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items. “Consisting essentially of” when used to define systems, compositions and methods, shall mean excluding other elements of any essential significance to the combination for the intended use. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for using the systems of the present disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

The term “sample” as used herein, includes blood, serum, plasma, urine, cerebrospinal fluid, saliva, derivatives of blood, serum, plasma, urine, cerebrospinal fluid, saliva, tears and any other body fluid that includes immunoglobulins.

By “isolated” is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type. The term “isolated” with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.

As used herein, the term “a part” or “affrontment” of a protein or polypeptide refers to a peptide or polypeptide comprising an amino acid sequence of at least 2 contiguous amino acid residues, at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 100 amino acid residues, at least 150 amino acid residues, or at least 200 amino acid residues of the amino acid sequence of a full-length peptide, polypeptide or protein.

Overview

The present disclosure relates to methods involving the agglutination of red blood cells (RBCs) or particles made of polymers such as latex and/or polystyrene, induced by specific antigen-antibody interaction that afford a highly sensitive and accurate assay for SARS-CoV antibodies, including SARS-CoV-2 antibodies. The methods of this disclosure are rapid and highly specific and sensitive.

In embodiments, there is provided a rapid, highly specific and sensitive assay that demonstrates the interaction between an antibody and its antigen. The assay may take no more than about 5 minutes. This interaction may be shown in antibodies against the severe acute respiratory syndrome coronavirus (SARS-CoV) including SARS-CoV-2 and proteins produced by vaccines.

The particle agglutination antibody assay of the present disclosure has been validated using 169 plasma samples that were tested positive for COVID-19 by PCR, 121 samples that were PCR negative and 100 SARS-CoV-2 naive plasma samples. The agglutination-based antibody assay produced 100% specificity and -98% sensitivity. Importantly, it detected antibodies in 92% COVID-19 patients on the day of diagnosis, rivaling the sensitivity of many PCR (swab test) and ELISA (antibody) tests. Second, the agglutination test is fast. It takes about two minutes from mixing the plasma with the latex particle to getting the result. Third, the antibody test is simple and requires no specific instrument. As the formation of the clumps by the latex beads is easy to identify, the test may be performed by an average person with no special training. Because of the few (if any) instruments needed, the assay is ideal for POC antibody. Fourth, because the test only takes a small amount of plasma, for example about 5 μl plasma/serum, and because the test is compatible with whole blood, it may be possible to develop the agglutination assay into a finger prick blood test. Fifth, the agglutination test can be modified to detect neutralizing antibodies to the virus when it is performed in the presence of ACE2 (angiotensin converting enzyme 2) which mediates virus entry into host cells¹⁴. Sixth, the agglutination assay may be used to detect total antibody or neutralizing antibody titer in a semi-quantitative manner. The syringe gel invention of ours will provide a semi-quantitative test within minutes without special instrumentation. Seventh, small epitope antigen peptides of no more than 25, 20,15, or 10 amino acid residues in length can be used to effectively detect SARS- CoV-2 in plasma, serum, or whole blood samples. An antigen peptide may be used alone or in combination with one or more other antigen peptides from the same or a different protein of the SARS-CoV2 virus. When epitopes obtained from Variants of Interests (VOC) are used, the agglutination assays and syringe system of the present invention serve to test VOC specific antibody responses. Finally, the low cost of the bead agglutination assay makes it affordable and ideal for antibody testing for large populations. The tests of the present disclosure are suitable for detection of SARS- CoV-2 antibodies in any fluid, aqueous sample. Most commonly, it is applied to blood, blood serum or plasma (including derivatives thereof), but it can also be applied to other fluid samples, such as, urine, cerebrospinal fluid and saliva. The tests of present disclosure are also suitable for detection antibodies to specific antigens in general. Taken together, the agglutination-based antibody tests require no instrument and accurately generate results typically in about 2 minutes. These tests can be used in point-of-care settings or at-home.

The present invention is applicable to agglutination immunoassays based on red blood cells or based on a variety of latex particles, polystyrene microspheres or particles made of rubber or other suitable polymers other than latex and polystyrene. Most latexes are composed of particles having a net negative surface charge at neutral pH. As used herein, the term latex is intended to mean the property of suspension of discrete microparticles in an aqueous liquid.

Latex particles useful in the present embodiment will be evident to the worker familiar with the field of latex agglutination immunoassay. In general, such particles require the properties necessary to serve as a stable support for the desired antibody antigen for the assay and to undergo agglutination in the presence of an antibody against SARS-CoV2 sufficient for analytical purposes. Latex particles are prepared generally by emulsion polymerization or suspension polymerization [Bangs, L.B. (1984) Uniform Latex Particles, Seragen Diagnostics Inc., Indianapolis, Ind., USA],

Swollen emulsion polymerization can also be used [Ugelstad, J. et al (1980) Adv. Colloid and Interface Sci. 13:101-140]. A good selection of latex particles are commercially available. Polystyrene particles are particularly useful.

As such, in one embodiment, the present disclosure relates to a method for detecting a humoral immune response to SARS-CoV in a subject, comprising the steps of: (a) contacting a sample from said subject with particles covered with a SARS- CoV antigen; (b) allowing the particles covered with the SARS-CoV antigen to agglutinate, and (c) detecting occurrence of agglutination, wherein occurrence of agglutination of the particles covered with the SARS-CoV antigen indicates a positive humoral immune response to SARS-CoV in the subject.

In another embodiment, the present disclosure relates to a method of measuring the level of immunoglobulins against a SARS-CoV in a sample from a subject, comprising the steps of: (a) contacting the sample with particles covered with a SARS- CoV antigen under conditions that allow the particles covered with the SARS-CoV antigen to agglutinate in the presence of immunoglobulins against SARS-CoV; and (b) detecting occurrence or absence of particle agglutination in the sample, said occurrence or absence of particle agglutination in the sample having an agglutination area, and correlating the agglutination area of the sample with the agglutination areas of multiple control agglutinations, each control agglutination area containing known concentrations of immunoglobulins against SARS-CoV, to provide a measure of the level of the immunoglobulins against the SARS-CoV in the sample.

In another embodiment, the present disclosure provides for a method for semi- quantitatively measure of a titer of neutralizing antibody against a SARS-CoV in a subject, comprising the steps of: (a) contacting a sample from said subject with particles covered with a SARS-CoV antigen; (b) allowing the particles covered with the SARS-CoV-2 antigen to agglutinate into a clump area; (c) calculating the percentage of agglutination based on agglutination/clumps area relative to the total particle reaction area, and (d) plotting the percentage of agglutination against an antibody titer curve to obtain the titer of neutralizing antibody against the SARS-CoV in the subject.

The assays of the present disclosure also allow quantitative, semi-quantitative and qualitative detection of SARS-CoV-2 antibodies in a sample and can be used to determine whether an individual produces antibody in response to infection or as a result of vaccine administration.

Detecting and/or Measuring Neutralizing Antibodies

In another embodiment, the present invention provides for a method to detect and/or measure neutralization antibodies. With reference to the top schematic graph of Fig. 11 , the particles of the present invention coated with S-RBD can be used to detect/measure total anti-SARS-CoV-2 antibodies. In the absence of recombinant ACE2 (angiotensin converting enzyme 2), the agglutination of the S-RBD-coated beads (i.e., the agglutination score) measures the total amount of antibody present in the fluid sample (upper panel). In contrast, with reference to the bottom schematic graph of Fig. 11 , when ACE2 is preincubated with the S-RBD-beads, the RBD epitopes will be masked by the bound ACE2. This results in the reduction of agglutination induced by the neutralization antibodies (denoted in Y symbol having an arrow), but not by the non-neutralizing antibodies (denoted with the Y symbol). As illustrated in Fig. 11 , neutralization antibody (Nab) titer determined using the bead agglutination (+/-ACE2) method correlated significantly with the Nab titer determined using the ELISA-based method (as illustrated in Fig. 10).

As such, in another embodiment, the present invention relates to a method of detecting presence of SARS-CoV (including SARS-CoV-2) neutralizing immunoglobulins in a sample taken from a subject, the sample having immunoglobulins, comprising: (a) contacting/mixing a portion or aliquot of the sample taken from the subject with particles covered with the SARS-CoV receptor binding domain (RBD) of the S protein (S-RBD) under conditions that allow the particles covered with the S-RBD to agglutinate in the presence of immunoglobulins against the S-RBD (agglutination conditions) and obtaining a first agglutination score (i.e., the level or intensity of agglutination), (b) mixing particles covered with the S-RBD with ACE2 to form a mixture, and (c) adding the mixture to another portion or aliquot of the sample taken from the subject under the same agglutination conditions as in step (a), and obtaining a second agglutination score. A reduction in the first agglutination score of the sample in step (a) relative to the second agglutination score of the ACE2 mixture in step (c) is indicative of the presence of the SARS-CoV neutralizing immunoglobulins in the sample taken from subject. In aspect, the S-RBD is the S-RBD of SARS-CoV, including SARS-CoV-2. In another aspect, the method further comprises comparing the first agglutination score and the second agglutination score with the agglutination scores of control samples having known amounts of neutralizing immunoglobulins, thereby providing a measurement of the neutralizing immunoglobulins in the sample based on said comparison.

Detecting and/or Measuring Antibodies by Syringe Gel Agglutination

In another embodiment, the present invention provides for a semi-quantitative method to measure SARS-CoV-2 antibody by syringe gel agglutination.

With reference to Figs. 14A, in a first step (1), a sample taken from a subject (e.g., finger prick blood, plasma, serum, urine, cerebrospinal fluid, saliva, tears and so forth) is diluted in a buffer (which lyse the red blood cells in the case when whole blood is used in the assay) and mixed with or contacted with antigen (including epitope)-coated dyed beads (in this particular case the beads are red-dyed). The mixture is allowed to stand in room temperature (for example for about 5 minutes). In Step (2), the mixture of sample with antigen-coated beads is transferred to a syringe pre-filled with hydrogels or polymers with pore size (such as from about 1 μm to about 100 μm) suitable for separating the agglutinated from the non-agglutinated beads. The mixture may be added from the top or side or the syringe or drawn from the bottom of the syringe. In Step (3), the plunger of the syringe is inserted and pushed (if the mixture is added to the top of the gel/polymer) or pulled (if the mixture is drawn from the bottom of the gel/polymer) for a certain distance determined by the amount of sample/buffer used and the length of the syringe gel. This pushing or pulling (as the case may be) of the plunger will result in the separation of the agglutinated beads (which cannot go through the gel/polymer) from the non-agglutinated beads (which are capable of penetrating the gel/polymer). The distance of plunger pushing or pulling may be standardized using antigen coated beads mixed with a certain amount of pure IgG protein (specific to the coated antigen) that produce the aggregation scores 0-4 (as in Fig. 4A or Fig. 13). The distance should be sufficient to push the beads with a score

“0” to the bottom end of the tube/column gel yet allow separation of samples with the scores 1 to 4. To facilitate semi-quantitative antibody determination using the syringe gel method, the positions of the agglutinated bead band corresponding to the scores 1 , 2, 3, 4 will be standardized using pure IgG that produces approximately 25%, 50%, 75% and 100% agglutination, respectively based on the agglutination assay described for Fig. 4. The position of the band of beads provides a semi-quantitative measurement of antibody titer similar to that in the gel-card assay (see Fig. 13). Fig. 14B provides photographs showing an example on how the syringe gel assay worked for a control sample (left) without anti-S antibody and a SARS-CoV-2+ sample with strong (4+) anti-S (spike) antibody response.

In one embodiment, a method to detect immunoglobulins against a target antigen in a subject comprises: (a) mixing a sample taken from the subject with dyed particles coated with the target antigen (“non-agglutinated particles”) to form a mixture, such that when the sample contains anti-target antigen immunoglobulins, the dyed particles coated with the target antigen and the anti-target antigen immunoglobulins form agglutinated particles, (b) loading the mixture to one end of a tube pre-loaded with a neutral hydrogel having a pore size for separating the dyed beads coated with the target antigen when agglutinated from dyed beads coated with the target antigen when non-agglutinated, and (c) pushing the mixture at the one end of the tube loaded with the neutral hydrogel towards a second end of the tube, wherein the agglutinated particles form a band of agglutinated particles, thereby detecting the immunoglobulins against the target antigen.

The target antigen may be an antigen from any pathogen of interest, including bacteria or virus that cause human or animal diseases, including HIV/AIDS, Ebola, influenza, Herpes, human papillomavirus, Espstein-Barr virus, hepatitis viruses, polio, rabies, meningitis, etc. In embodiments, the target antigen is a SARS-CoV antigen. In further embodiments, the target antigen is a SARS-CoV-2 antigen. In further embodiments, the target antigen is a protein or part of a protein of the disease-causing virus or bacterium.

SARS-CoV-2 Epitopes

In another embodiment, the present invention provides for specific epitopes that can be used in any of antibody tests or methods suitable to detect SARS-CoV-2 antibodies in a sample taken from an individual. Suitable antibody tests for detecting SARS-CoV-2 antibodies include the agglutination methods of the present invention, in which particles (red blood cells, latex particles, polystyrene microspheres, nanoparticles, rubber particles, non-rubber particles and so forth) are covered with the specific epitopes of the present invention. In addition, the specific epitopes of the present invention can be used in enzyme-linked immunosorbent assay (ELISA).

In embodiments, the particles are covered with one or with a combination of two or more of the specific epitopes of the present invention.

In embodiments, the specific epitopes of the present invention are from a SARS- CoV-2 antigens. In embodiments, the specific epitopes are from SARS-CoV-2 S protein. In embodiments, the specific epitopes are from the N protein of SARS-CoV- 2. In embodiments, the specific epitopes are from the M protein of SARS-CoV-2. In embodiments, the specific SARS-CoV-2 epitopes of the present invention are listed in Table 3. Table 4 lists Variant of Concern (VOC) from the Spike (S) protein of SARS- CoV-2. Table 5 provides for the sequence alignment for the spike (S) protein from SARS-CoV (SEQ ID NO: 228) and SARS-CoV-2 (SEQ ID NO: 229). Table 6 provides the sequence alignment for the nucleocapsid (N) protein from SARS-CoV (SEQ ID NO: 230) and SARS-CoV-2 (SEQ ID NO: 231).

As such, in one embodiment, the present invention provides a method of detecting the presence or absence of a humoral immune response to SARS-CoV-2, a variant of SARS-CoV-2, or a vaccine against SARS-CoV-2, including vaccines based on Spike mRNA, in a subject, comprising the steps of: (a) contacting a sample with antibodies/immunoglobulins from said subject with one SARS-CoV-2 epitope selected from Table 3 or with a combination of two or more of the SARS-CoV-2 epitopes selected from the epitopes listed in Table 3; (b) detecting the presence or absence of binding of the antibodies/immunoglobulins to said one SARS-CoV-2 epitope selected from Table 3 or combination of two or more of the epitopes selected Table 3, wherein detecting binding of the antibodies to said one SARS-CoV-2 epitope selected from Table 3 or the combination of two or more SARS-CoV-2 epitopes selected from Table 3 is indicative of a humoral response to SARS-CoV-2. In embodiments, the sample is blood, serum, plasma, cerebrospinal fluid, saliva, urine, saliva, tears or any other fluid sample taken from a subject that contains immunoglobulins.

In embodiments, step (a) comprises contacting the sample with particles covered with the one SARS-CoV-2 epitope selected from Table 3 or with the combination of two or more of the SARS-CoV-2 epitopes listed in Table 3, and step (b) comprises detecting occurrence of agglutination of the particles covered with the one SARS-CoV- 2 epitope or with the combination of two or more SARS-CoV-2 epitopes, wherein occurrence of agglutination of the particles covered with the one SARS-CoV epitope or with the combination of two or more SARS-CoV-2 epitopes indicates a positive humoral immune response to SARS-CoV-2, a variant of SARS-CoV-2, or a SARS- CoV-2 vaccine in the subject.

In embodiments, the present invention provides for an isolated epitope or recombinant polypeptide, wherein said isolated epitope or recombinant polypeptide binds to a SARS-CoV-2 antibody or immunoglobulin, wherein said epitope or recombinant polypeptide are selected from SEQ ID NO: 1 to 227.

In order to aid in the understanding and preparation of the present disclosure, the following illustrative, non-limiting examples are provided.

EXAMPLES Blood sample collection

Blood samples were collected following a protocol (study number: 116284) approved by the Research Ethics Board (REB) of Western University (London, Canada). The plasma samples were de-identified prior to transfer from the Laboratory of Clinical Medicine (London Health Sciences Center, London, Canada) to a biosafety Level 3 (CL3) lab (ImPaKT, Western University) following Transportation of Dangerous Goods

(TDG) guidelines. All plasma samples were heat-inactivated at 56 °C for 30 minutes at the ImPaKT CL3 facility as per Western university biosafety regulation. Heat inactivated plasma samples were then transferred to the testing laboratory. We tested the effect of heat inactivation on SARS-CoV-2 antibody titer and found no significant impact of heat-inactivation.

Recombinant proteins and peptide antigens

Protein antigens such as the spike receptor binding domain (S-RBD) and the nucleocapsid (N) may be produced in mammalian or bacterial cells by recombinant technologies or purchased from any vendor that sells these proteins as products. For example, S-RBD was obtained from ThermoFisher Scientific (RP-87678) and nucleocapsid from RayBiotech (230-01104). The RNA-binding domain of the nucleocapsid (N-RBD) was cloned into the pMCSG53 (residues 47-173, containing a 6xHis tag and a Tobacco Etch Virus (TEV) cleavage site) prokaryotic expression vector and expressed in E. coli. The protein was purified by Ni-NTA chromatography. The expression plasmid for human ACE2 (residues 1-615) was cloned into the mammalian expression vector paH (containing an 8XHis tag) and the protein was produced by transient transfection of Expi293F cells (ThermoFisher Scientific, Cat# A145527). Supernatant from the transfected cells was harvested 96 hours post- transfection and the protein was purified by Ni-NTA chromatography. Protein purity was confirmed by SDS-PAGE (Fig. 9).

The peptide antigens were synthesized by the solid phase peptide synthesis technique based on Fmoc/HBTU chemistry. Preparation of SARS-CoV-2 antigen coated latex particles

Blue dyed polystyrene latex beads, 0.8 μm in diameter, were purchased from Sigma Aldrich 325 (L1398). Prior to use, the latex beads were washed according to the manufacturer’s instructions with some modifications. Briefly, 2.5ml_ of 5% (w/v) latex suspension was washed twice in 10 mL PBS buffer (135 mM NaCI, 2.6 mM KCI, 8 mM Na2HP04, and 1.5 mM Na2HP04, pH 7.4) by mixing and centrifuging the latex suspension at 3,000g for 10 minutes at room temperature. The beads were then resuspended with 2.5 ml 0.025M MES buffer (2-(N-Morpholino) ethanesulfonic acid, pH 6.0) to obtain 5% (w/v) suspension. SARS-CoV-2 antigen-latex particle conjugates were prepared by passive adsorption following the procedures described by Mahat et al¹⁵, with some modifications. Briefly, 0.4 mL of 5% (w/v) latex suspension was centrifuged at 3,000 g for 5 minutes at room temperature, and the supernatant was discarded. The beads were incubated with 200 μg recombinant Receptor Binding Domain of the SARS-CoV-2 spike protein (S-RBD) (Structural Genomics Consortium, University of Toronto) or the Nucleocapsid protein (N protein) (RayBiotech, 230-01104) in 4 mL MES buffer. The mixture was allowed to incubate for 24 hours at 4°C with periodic mixing. After conjugation, the antigen-latex bead conjugate was centrifuged, and the supernatant was kept for determination of unabsorbed protein concentration (Bio-Rad protein assay kit). The antigen-bead conjugate was washed twice with PBS and blocked for 30 min at room temperature in PBS containing 3% bovine serum albumin (BSA). The conjugate was then resuspended at 2.5% (w/v) in PBS containing 1 % BSA and stored at 4°C until use.

Preparation of SARS-CoV-2 peptide antigen conjugated latex particles and peptide antigen-based agglutination assay

Blue dyed carboxylate-modified, streptavidin-polystyrene latex beads, 0.25 μm in diameter, were purchased from Sigma Aldrich (L6155). Carboxylate-modified latex- streptavidin beads were suspended at 2.5% (w/v) using assay buffer, 0.025M MES- Tween 20 buffer (2-(N-Morpholino) ethanesulfonic acid, 0.05% pH 6.0). Synthetic biotin-labeled SARS-CoV2 peptides (a single epitope peptide or a mixture of two different epitope peptides in 1 : 1 molar ratio) were suspended in the same assay buffer at the concentration 500μg/ml. The biotin-peptides were incubated with streptavidin- latex beads for 1 hour at room temperature. The epitope peptide conjugated latex beads complex were washed twice with PBS buffer (135 mM NaCI, 2.6 mM KCI, 8 mM Na2HP04, and 1.5 mM KH2P04, pH 7.4) by mixing and centrifuging the latex suspension at 5,000g for 10 min. The peptide antigen-bead conjugate was blocked for 30 min at room temperature in PBS containing 3% bovine serum albumin (BSA). The conjugate was then resuspended at 2.5% (w/v) in PBS containing 1% BSA and stored at 4°C until use. For the agglutination assay, 5 μl plasma was mixed with 25 mI peptide- conjugated latex beads (2.5%, w/v) per assay as described in the full protein antigen agglutination assay.

Agglutination assay for SARS-CoV-2 antibody testing and data interpretation

For the agglutination assay, 5 μl plasma was mixed with 25 mI antigen-coated beads (2.5%, w/v) per assay. The agglutination was allowed to proceed for 2 min at room temperature before imaging with a camera. The relative degree of agglutination induced by the SARS-CoV-2 antibody was measured by the area of clump formation based on the corresponding image. Agglutination data analyses were performed using qualitative and semi-quantitative assessments. For semi-quantification of agglutination, the image analysis software Qupath (vO.1.2) was used (https://qupath.github.io/) and quantification was done by calculating the percentage of agglutination based on estimated agglutination/clumps area (mm²) relative to the total latex reaction area. In qualitative assessments, agglutination intensity was inspected visually, and agglutination score was assigned (i.e. , 1 , 2, 3 and 4). Specifically, 1 corresponds to small clumps with -25% agglutination, 2 (-50% agglutination), 3 (-75% agglutination), and 4 (large clumps that forms in less than 1 min with -100% agglutination). Based on data from the COVID-19 negative samples (including NAAT negative and samples collected in 2018), the cut-off for positivity was set to 5% of agglutination. Preparation of red blood cells conjugated with SARS-CoV-2 antigen

The recombinant spike receptor-binding domain (S-RBD) or the nucleocapsid RNA- binding domain (N-RBD) was conjugated in 30-fold molar excess biotin using EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Scientific, A35358). Excess unbound biotin was removed using ZebaTM Spin Desalting Columns, 7KMWCO (Thermo Scientific, 89890). Anti-D-lgG was purified from Immucor Anti-D Series 4 (IgG & IgM monoclonal blend) by using protein A magnetic affinity purification (G8782, Promega). The purified anti-D-lgG was then concentrated (3mg/ml) and stored at 4 °C until use. Anti-D was then conjugated with streptavidin according to manufacturer instruction (ab102921 , abeam). Bioconjugation of Anti-D-lgG-streptavidin with Reagent Red Blood Cells (RRBC) [0.8% R2R2; blood group O; Rh/D-antigen+] (Ortho-Clinical Diagnostics

SELECTOGEN, 6902315) was done by incubating the anti-D-lgG-streptavidin with RRBC for 30 min at room temperature. The RRBC-anti-D-streptavidin complex was then washed twice with low ionic strength RBC diluent (MTS™ Diluent 2 PLUS; Micro Typing Inc., MTS9330S). The complex was centrifuged at 1000g for 2 min to remove unbound anti-D-lgG streptavidin and was then resuspended in the same RBC diluent. RBC-anti-D-lgG-streptavidin was then conjugated with either biotin-S-RBD or biotin- N-RBD for 15 min at room temperature. The RRBC-anti-D-sterptaviding-biotin-S- RBD/N-RBD was stored at 4 °C until use. The RRBC agglutination assay was carried out in the same way as for latex agglutination described above.

Agglutination Assay using Polybeads carboxylate red-dyed microspheres coated with S-RBD

Red-dyed carboxylate polystyrene beads, 1 μm in diameter, were purchased from Polysciences Inc., (19119-15325). The Carboxylated Polybeads were covalently coupled to antigens using the carbodiimide method (PolyLink Protein Coupling Kit; 24350-1) with some modifications. Briefly, 0.5ml of 2.5% (w/v) Carboxylated Polybeads was washed twice in 1.5 mL 0.1 M Carbonate Buffer (0.1 M Na2C03 was added to 0.1 M NaHC03 until pH 9.6 is reached) by mixing and centrifuging the latex suspension at 500g for 5 minutes at room temperature. The Polybeads were then resuspended in 0.625ml of 0.1 M MES buffer [2-(N-Morpholino) ethanesulfonic acid, pH 6.0], Equal volume of carbodiimide (2%) was incubated with the beads for 15 min at room temperature. The carbodiimide coupled beads (carbodiimide-beads) were then washed twice with 0.1 M MES buffer. The beads were then resuspended in 1 ,2ml of 0.2M borate buffer (0.2M boric acid, 1 M NaOH, pH 8.5). Next, 200 μg S-RBD protein was incubated with carbodiimide-beads on an end-to-end mixer (overnight at room temperature). S-RBD-beads were washed twice with 1 ml of 0.2M borate buffer. To block unreacted sites on the beads, 50pl of 0.25 M ethanolamine was added to the S-RBD-beads and incubated for 30 minutes at room temperature. To block non- specific protein binding sites, S-RBD-beads were then suspended in 1ml of blocking buffer (10mg/ml BSA solution in 0.2 M borate buffer) for 30 minutes at room temperature. The S-RBD-beads were then stored in 0.5ml of Storage Buffer [0.01 M phosphate buffer (pH 7.4), 1% BSA, 0.1% sodium azide and 5% glycerol] at 4°C until use. The Polybeads agglutination assay was carried out in the same way as for the latex agglutination assay described above.

S-RBD-ACE2 binding ELISA and surrogate neutralization assay

ELISA plate Coating and blocking -S-RBD was dissolved (5 μg/ml) in T ris buffer saline (TBS) (20 mM Tris, 150 mM NaCI, pH7.4) and 100 μl of the S-RBD solution was added to each well of an ELISA plate and incubate at 4°C overnight with slow shaking. The antigen-coated wells were washed 3 times with TBS-tween (TBST) (20 mM Tris, 150 mM NaCI, 0.1% Tween 20). The S-RBD coated wells were blocked by 100 mI of the ChonBlock™ blocking/sample dilution ELISA buffer (Chondrex, Inc., 9068) for 1 hour at room temperature with slow shaking followed washing 3 times with TBST.

ACE2:S-RBD binding assay- ACE2 was biotinylated as described above. Biotin-ACE2 (1 μg/ml) was added to S-RBD-coated plate after blocking and incubated for 1 hour at room temperature. The wells were washed 3 times with TBST to remove unbound biotin-ACE2. Streptavidin-HRP (1000-fold dilution with Chonblock blocking buffer) was then added to each well and incubated for 1 hour at room temperature. The wells were washed 3 times with TBST and TMB substrate (3,3',5,5'-Tetramethylbenzidine, Thermo Scientific, N301 ) was added for reaction development and 0.18 M H₂SO₄ was used to stop reaction. Absorbance at 450nm was measured to detect the S-RBD bound ACE2.

SARS-CoV-2 antibody neutralization assay- Plasma was diluted 1 :100 and incubated with S-RBD-coated wells (blocked) for 1hour at room temperature. The wells were washed three times with TBST. Biotin-ACE2 was then added to the wells and incubated for 1 hour at room temperature followed by washing, reaction development and detection as described above.

Neutralization antibody assay by agglutination Step I: Measuring total Anti-S-RBD Antibodies (Neutralizing antibodies plus Non-Neutralizing antibodies)-Latex beads or Polybeads carboxylate were conjugated with S-RBD as described above. Agglutination assay was done by mixing 5 mI plasma with 25 mI S-RBD-coated beads (2.5%, w/v) per assay. The agglutination was allowed to proceed for 2 min at room temperature before imaging with a camera. The relative degree of agglutination induced by the S-RBD antibody was measured by the area of clump formation based on the corresponding image. Agglutination data analyses were performed using qualitative and semi-quantitative assessments as detailed above. The agglutination percentage was calculated and was assigned as the total Anti-S-RBD antibody (%).

Step II: Measuring Neutralizing Antibodies (Total Anti-S-RBD - Non-Neutralizing anti-S-RBD) - Using the same patient plasma tested in step I, agglutination assay was repeated with modification. Briefly, 25mI of ACE2 (5μg/ml) was added to 25mI of S- RBD-coated beads and incubated for 10 min at room temperature to block all neutralizing antibodies binding sites on the S-RBD. Blocking of ACE2 binding sites was assessed using biotin-ACE2-streptavidin to determine the suitable concentration and ensure that the added ACE2 (25 ul of 5μg/ml) was sufficient to block ACE2/Nab binding site on the S-RBD-coated beads. Then 10mI of the same plasma as in Step I was added to 50mI S-RBD-coated beads-ACE2 mix. The agglutination was allowed to proceed for 2 min at room temperature before imaging with a camera. The relative degree of agglutination induced by the S-RBD antibody was measured by the area of clump formation based on the corresponding image. The agglutination percentage was calculated and was assigned as the non-Neutralizing Anti-S-RBD antibody (%).

Neutralization antibody (Nab) calculation: The percentage of Neutralizing anti-S-RBD antibody was calculating using the following equation: Neutralizing Anti-S-RBD (%) - [Total Anti-S-RBD (%, calculated in Step I) - non-Neutralizing Anti-S-RBD(%, calculated in Step II)].

Gel card agglutination assay Agglutination assay was done using the DG Gel cards/microtubes (Ortho-Clinical Diagnostics, Cat#: MTS085014). Briefly, the foil seal was removed from the individual microtubes to be used for testing. 5mI plasma or whole blood was mixed with 10mI SARS-CoV2 antigen/epitopes-coated beads as described before. Then the mixture was loaded to the Gel microtubes then centrifuged at 500g for 10min. After centrifugation, the gel card was removed from the centrifuge the results were read.

Results interpretation of gel card agglutination

Negative: Appears as non-agglutinated beads at the bottom of the gel column and no visible agglutinated beads in the rest of the gel column. Positive 1+: Appears as small-sized clumps of agglutinated beads most frequently in the lower half of the gel column. A small pellet may also be observed at the bottom of the gel column.

Positive 2+: Appears as small or medium-sized clumps of agglutinated beads throughout the gel column. A few unagglutinated beads may be visible at the bottom of the gel column.

Positive 3+: Appears as medium-sized clumps of agglutinated beads in the upper half of the gel column.

Positive 4+: Appears as a well-defined band of agglutinated beads in the top part gel column. A few agglutinated beads may be visible below the band. Syringe gel antibody test

We have designed a syringe-like apparatus that is packed with neutral gel (such as DG gel used in gel card agglutination) or any other gel (agarose or acrylamide based) with a pore size that can separate the non-agglutinated beads from the agglutinated beads (e.g., 1-100 μm in diameter). The side of the barrel has a small opening that allow the addition of the sample from the agglutination assay and the opening is capped after the agglutination assay sample is added (Fig. 14A). In the proof-of- concept experiment, we simplified the syringe gel test using 1 ml syringe. The barrel/tube of a 1 ml syringe was packed with 200 μl NeutralGel (Fig. 14B). The agglutination assay was done by mixing 5mI plasma or whole blood with 10mI SARS- CoV2 antigen/epitope-coated beads as described previously. Then the beads- plasma/blood mixture was loaded to the top of the gel in the syringe barrel. The agglutination was allowed to proceed for 2 min then the syringe plunger was inserted and pushed gently down for a predetermined distance. This new apparatus (or its variations) and method of detecting and/or measuring antibodies in a sample in the syringe gel serves to replace the centrifugation-based method in the gel card agglutination antibody test. Results interpretation of syringe gel agglutination

Negative: Appears as non-agglutinated beads at the bottom of the gel syringe and no visible agglutinated beads in the rest of the gel syringe.

Positive 1+: Appears as small-sized clumps of agglutinated beads most frequently in the lower half of the gel syringe. A small pellet may also be observed at the bottom of the gel syringe.

Positive 2+: Appears as small or medium-sized clumps of agglutinated beads throughout the gel syringe. A few unagglutinated beads may be visible at the bottom of the gel syringe.

Positive 3+: Appears as medium-sized clumps of agglutinated beads in the upper half of the gel syringe.

Positive 4+: Appears as a well-defined band of agglutinated beads in the top part gel syringe. A few agglutinated beads may be visible below the band.

Statistical analysis

All statistical analyses were done using the GraphPad Prism9 software. Specifically, the Hill coefficient (h) was calculated from fitting agglutination data obtained using anti- S-RBD and anti-N antibodies to the Hill equation. COVID-19+ samples with distinct agglutination scores and COVID-19- samples were analyzed using unpaired 412 t-test with Welch's correction (no assumption of equal standard deviation between two groups). Changes in agglutination for samples before and after heat-inactivation were analyzed by paired t-test. Spearman’s correlation rank was done to study correlation between antibody titter and ACE2:S-RBD neutralization efficiency. RESULTS

Agglutination-based serologic testing for SARS-CoV-2 antibodies

Group O (R2R2) RBCs carrying the D antigen were labeled with the spike RBD (S- RBD) or the RNA binding domain of the nucleocapsid (N-RBD) protein through streptavidin-biotin mediated coupling (Figs. 6A-6B, see Materials and Methods for details). Incubating the antigen-coated RBCs with COVID-19⁺ plasma led to robust agglutination whereas the COVID-19- plasma failed to induce RBC agglutination. Therefore, aggregation of the S-RBD/N-RBD-coated RBCs can be used to detect antibody response to SARS-CoV-2 (Figs. 6A-6B).

For a cost-effective agglutination assay, latex particles were coated with recombinant Spike (S) or Nucleocapsid (N) protein or a fragment of either protein (including the RBD) of SARS-CoV-2 (Fig. 1). The antigen coated latex beads were first tested with a monoclonal anti-S-RBD and a polyclonal anti-nucleocapsid antibody. Upon incubating with the corresponding antibody, the antigen-coated latex particles formed clumps within two minutes. Importantly, the area of clump formation grew larger with increasing antibody concentrations (Fig. 2). Although latex agglutination was commonly used as a qualitative assay, it is possible to determine the degree of agglutination based on the area of clump formation via image analysis. As shown in Fig. 2, the percentage of agglutination for both the S-RBD- and N-coated latex particles increased when an incremental amount of anti-S-RBD or anti-N antibody was added. Fitting the data to Hill’s equation yielded Hill’s coefficient of 1 .7 for the former and 1.8 for the latter. Therefore, the antibody-induced agglutination of latex particles is a cooperative event (Figs. 2A-2B). We next examined if the latex agglutination assay could be used to gauge COVID-19 antibody response. Using plasma samples from patients tested positive for SARS- CoV-2 by NAAT (nucleic acid amplification test) and confirmed for strong antibody response by ELISA, we found that the patient plasma samples were not only capable of inducing agglutination of the S-RBD- or N-coated latex particles, they did so in a concentration-dependent manner. As shown in Figs. 2C-2D, the extent of agglutination (or agglutination score) decreased as the plasma was diluted, indicating that the agglutination assay may be used to estimate antibody titer as in an ELISA-based antibody test. The latex agglutination-based antibody assay showed high sensitivity and specificity

To validate the antibody test based on latex particle agglutination, we carried out agglutination assays for 290 residual plasma samples from individuals that were tested positive (169) or negative (121) for virus RNA by the Roche cobas SARS-CoV-2 test⁹. To assess specificity, we also included 100 virus-naive samples banked in 2018 in our agglutination assay. None of the 121 SARS-CoV-2- or the 100 Pre-COVID-19 plasma samples was capable of promoting the agglutination of either the S-RBD- or N-coated latex particles, indicating 100% specificity for the agglutination assay (Table 1). In contrast, of the 169 SARS-CoV-2⁺ plasma samples tested, 166 (98.2%) promoted agglutination in response to the S-RBD antigen and 164 (97%) to the N antigen, with overall sensitivity of 98.2%. We compared the latex agglutination assay with the Euroimmune IgG test for the S antibody and the Roche Elecsys® Total assay for the N antibody using the same set of SARS-CoV-2⁺ plasma samples and found that the latex agglutination assay outperformed both ELISA-based antibody tests (Table 2). The agglutination assay of the present disclosure also exhibited better specificity than either ELISA kit (Table 2). Quantification of the agglutination data showed that the COVID-19⁺ group is significantly different from the COVID-19- or pre-COVID-19 group, indicating that the latex agglutination assay effectively distinguished SARS-CoV2⁺ from SARS-COV2- individuals (Figs. 3A-3B). Based on the background signals of the COVID-19- samples (0-4% agglutination for both S-RBD- and N-coated latex particles), we set 5% agglutination as the cut-off for antibody positivity. To facilitate the use of the latex agglutination assay as a simple, semi-quantitative antibody test, we developed a numerical scoring system for antibody response. The agglutination score may be readily assigned by visual inspection and comparing to reference wells containing a predetermined amount of pure anti-S-RBD or anti-N antibody (Fig. 4A). We assigned the scores 1 , 2, 3 and 4 to samples that produced 5-25%, 25-50%, 50-75% and >75% agglutination, respectively (Fig. 4B). We found that this scoring scheme effectively distinguished samples with strong antibody response from those with medium or weak ones (Figs. 5A-5B).

Agglutination assay using Polybeads carboxylate red-dyed microspheres and its application in determining antibody response to vaccination

The same principle of using latex particle agglutination in response to antibody binding was tested on other carriers, including polystyrene microspheres (Polysciences). The Polybeads are functionalized with carboxylate on the surface, facilitating the covalent coupling of protein or epitope/peptide antigens. We found that the Polybeads microspheres (red-dyed) covalently coupled with the S-RBD antigen could be used in the agglutination assay for SARS-CoV-2 antibodies (Fig. 7).

The Polybeads were coated with either the Spike ectodomain or full-length (FL) Nucleocapsid (N) and the resulting antigen-coated beads were used to determine the S- and N-antibody responses in individuals 4 weeks after vaccination with the BNT162B2 vaccine or hospitalized patients tested positive for SARS-CoV-2. The vaccinated individuals (whose status of SARS-CoV-2 infection history is not known) had strong S antibodies, but no or low N antibodies. In contrast, the hospitalized patients showed strong antibodies to both S and N (Fig. 8).

An ACE2-RBD binding competition ELISA method to measure neutralizing antibody titer Because neutralizing antibodies (Nab) play a pivotal role in the humoral immune response to the virus¹⁶, we developed a surrogate neutralization assay by measuring the efficacy of patient plasma in blocking S-RBD binding to its host receptor, angiotensin converting enzyme 2 (ACE2) in vitro using purified proteins (Fig. 10). Similar approaches have been used by others to evaluate neutralization efficiency of patient plasma or therapeutic antibodies¹⁷’¹⁸. Briefly, binding of biotinylated ACE2 to immobilized S-RBD is detected by ELISA through HRP-conjugated streptavidin. The presence of neutralizing antibody would block this interaction, resulting in reduction of the ELISA signal (Fig. 10A). Using this surrogate neutralization assay, we found that the neutralization efficiency increased with the agglutination score for the S-RBD antibody (Fig. 10B). Intriguingly, comparison of samples with distinct S-RBD and N antibody responses indicated that the neutralization efficiency was significantly correlated with the S-RBD, but not the N specific antibody strength (Fig. 10C).

An agglutination-based method for determining SARS-CoV-2 neutralizing antibody

By taking advantage of the competition between ACE2 and Nab for binding the S- RBD, we developed a new method to measure the Nab and Nab/total antibody ratio by performing the S-RBD-bead agglutination in the presence or absence of ACE2 (Fig. 11). The degree of agglutination of the S-RBD-coated beads when mixed with the plasma measures the total antibody response in the absence of ACE2. In contrast, when ACE2 is added prior to the addition of the plasma, the agglutination measures the non-neutralizing antibodies. Therefore, the neutralizing antibody titer Nab =%Aggregation (-ACE2) - %Aggregation (+ACE2) (Fig. 12A). We used this method to compare the total antibody vs the Nab for 9 COVID-19+ plasma (Fig. 12B) and compare the Nab levels measured by the agglutination assay or the competition ELISA (Fig. 10) and found that the results from the two methods correlate significantly (Fig. 12C).

A semi-quantitative gel card agglutination assay for measuring SARS-CoV-2 antibodies Gel card agglutination is commonly used for blood typing in clinical labs. We tested if the bead/microsphere agglutination assay may be combined with gel card to provide a semi-quantitative measurement for SARS-CoV-2 antibodies. The agglutination product of the S-RBD-coated Polybeads mixed with either a COVID-19+ or COVID- plasma sample was added to the top of a Neutral Gel card/microtube (Ortho-Clinical Diagnostics). Centrifugation of the Gel card at 500g for 10 min led to the separation of the agglutinated beads (on the top) from the non-agglutinated beads (at the bottom) of the Gel card (Fig. 13). The position of the band of beads provided a measure of antibody strength (with scores from 1+ to 4+, indicating increasing antibody strength).

A novel syringe gel agglutination assay for measuring SARS-CoV-2 antibodies

The Gel card agglutination assay requires specialized equipment (e.g., centrifuge) and can only be performed in a clinical lab setting. To enable point-of-care (POC) testing, we developed a simplified version in which the porous gel is contained in a tube such as the barrel of a syringe. The agglutination is carried out in a similar manner as in Fig. 1 or Fig. 7 and the agglutination product (i.e. , the blood-buffer-beads mix) is then transferred to the top of the gel inside of the tube. The plunger is then pushed for a predetermined distance to force the beads into the gel matrix (for convenience, the predetermined distance may be marked on the tube of the syringe). While the non- aggregated beads will be able to traverse the gel matrix to reach the bottom of the syringe, the agglutinated beads are too large to go through the gel and will form a band that remains on the top of the gel. The position of the bead band will provide a semi- quantitative measure of the antibody strength in a similar manner as in a conventional gel card assay as described in legends of Fig. 13 and Fig. 14. With reference to Fig. 14B, we showed that the syringe gel assay produced the expected band position for a SARS-CoV-2+ sample with 4+ anti-S antibody response (as measured by regular agglutination assay described in Fig. 4) and a SARS-CoV-2- control sample.

The agglutination assay allowed for early antibody detection and tracking of dynamic antibody response We noted that the agglutination assay detected antibody response in >92% plasma samples collected on the day of SARS-CoV2⁺ diagnosis by Nucleic Acid Amplification Tests (NAAT) and in 100% samples on day 2 and afterward (Table 2). This is in stark contrast to the 47% to 83% sensitivity for ELISA-based antibody tests on samples collected within 7 days of positive NAAT⁹ (Table 2). The superb sensitivity of the bead agglutination assay suggests that it may be used to detect antibody response in the early stage of virus infection and monitor its dynamic changes over time¹²’¹⁹’²⁰. The superb sensitivity and specificity of our bead agglutination assay for patient samples collected early in hospitalization suggests that it may be used to diagnose active infection in conjunction with NAAT. Combined antibody and RNA testing may increase the sensitivity of the latter. Moreover, the agglutination-based antibody test may be used to monitor the evolvement of humoral immune reaction in infected individuals over time or the duration of antibody responses in vaccinated individuals.

A rapid agglutination assay to gauge epitope-specific antibody response

The role of humoral immunity in COVID-19 is not fully understood owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome. Using SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients plasma samples to identify antigens and epitopes²¹. This enabled us to develop epitope arrays and epitope-specific agglutination assays to gauge antibody responses systematically and with high resolution.

While the epitope peptide array may be used to determine antibody specificity in a systematic manner, it is not suitable for point-of-care (POC) testing. Nevertheless, the identification of specific epitopes that are either common to the COVID-19 patients examined or unique to groups with distinct clinical severity or outcome prompted us to develop a rapid test based on these epitopes. Inspired by the principle of antibody- dependent red blood cell agglutination¹, we developed an epitope-dependent agglutination assay to detect epitope-specific antibody response. Specifically, latex beads were coated with streptavidin and conjugated to one or more biotinylated epitope peptides. Antibodies specific to the epitopes were found to induce the agglutination of the corresponding latex beads within minutes (Fig. 15A), with the area of agglutination serving as a proxy of antibody titer. In principle, the latex bead agglutination assay is more sensitive than the peptide array as it detects the total antibodies (including IgG, IgM and IgA) rather than a specific isotype. To develop an epitope test to replace the S and N antigens, we coated the latex beads with the most prominent S or N epitopes. Specifically, latex beads were coated with a mixture of the S-811 (SEQ ID NO: 82) and S-1146 (SEQ ID NO: 97) (2S) peptides to represent the S antigen or the N-156 (SEQ ID NO: 116) and N-361(SEQ ID NO: 139) (2N) peptides to represent the N antigen. When evaluated using plasma samples from individuals who tested positive (COVID+) or negative (COVID-) for the SARS-CoV-2 virus or samples from healthy donors collected in 2018 (PreCOVID), the 2S- and 2N-based agglutination assays effectively distinguish the COVID+ plasma from the COVID- or PreCOVID plasma (Fig. 15B).

Epitope-specific antibody response distinguishes COVID-19 disease severity and outcome

To determine if the epitope-dependent agglutination assay could differentiate the different patient groups with moderate or severe systems or with alive orfatal outcome, we coated the latex beads with the S epitope S-811 (SEQ ID NO: 82), S-881 (SEQ ID NO: 86) or S-551 (SEQ ID NO: 48) or the N epitope N-156 (SEQ ID NO: 116) or N- 361 (SEQ ID NO: 139) and performed agglutination assays on 10 patients/group. While no agglutination was observed for the COVID- plasma, the COVID+ plasma promoted the agglutination of the latex beads in an epitope-dependent manner. We found that the group with severe disease had significantly greater S-811- (SEQ ID NO: 82) and N-361 (SEQ ID NO:139) -specific antibody responses than that with moderate conditions. The reverse was found true for the N-156 (SEQ ID NO: 116) epitope (Fig. 16A-16C). Similarly, significant differences in the antibodies specific for the S-811 , S- 881 , S-551 and N-156 epitopes were observed between the alive and fatality groups (Fig. 16D-16G). Notably, a high level of S-811 (SEQ ID NO: 82)-dependent agglutination was strongly and significantly correlated with patient death whereas even a moderate level of S-551 -specific antibody response was correlated significantly with favorable outcome. These data identified a group of epitopes, including S-811 (SEQ ID NO: 82), S-881 (SEQ ID NO: 86), S-551 (SEQ ID NO: 48) and N156 (SEQ ID NO: 116), to which antibody responses correlated with clinical severity and outcome of the

COVID-19 disease.

Epitope-resolved antibody testing not only affords a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS- CoV-2, it may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern (VOC) and evaluate antibody protection against VOC in which the specific mutations (eg., P681 H/R) result in changes in epitope specificity and/or affinity. Table 3 provides a comprehensive list of linear epitopes found in the S, N and M proteins of SARS-CoV- 2. Table 4 provides a list of mutations/mutated epitopes associated with VOC.

Table 1 : Clinical performance of the agglutination-based antibody assay

Table 2: Comparison in sensitivity and specificity between the agglutination- based and ELISA-based antibody assays

Table 3. A List of Epitope Peptides (predicted or experimentally identified) from SARS-CoV-2

Epitopes from the Spike (S) protein

Underlined peptides were used in the Examples (see Figs. 14B, 15A, 15D, 15E and 15F)

Epitopes from the Nucleocapsid (N) protein

Underlined peptides were used in the Examples (Figs. 14B, 15B, 15C and 15G) Epitopes from the M protein

Table 4: A list of Variants of Concern (VOC) peptides (reported or experimentally confirmed) from SARS CoV-2

Variants of concern epitopes from the Spike (S) protein

Variants of concern epitopes from the Nucleocapsid (N) protein

Table 5. Sequence alignment for the spike (S) protein from SARS-CoV (SEQ ID NO: 228) and SARS-CoV-2 (SEQ ID NO: 229) (Epitope peptides determined for

SARS-CoV-2 are underlined)

Table 6 Sequence alignment for the nucleocapsid (N) protein from SARS-CoV (SEQ ID NO: 230) and SARS-CoV-2 (SEQ ID NO: 231) (Epitope peptides determined for SARS-CoV-2 are underlined)

The above disclosure generally describes the present invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation. Other variations and modifications of the invention are possible. As such modifications or variations are believed to be within the sphere and scope of the invention as defined by the claims appended hereto.

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17 Tortorici MA, B. M., Lempp FA, Pinto D, Dang HV, Rosen LE, McCallum M, Bowen J, Minola A, Jaconi S, Zatta F, De Marco A, Guarino B, Bianchi S, Lauron EJ, Tucker H, Zhou J, Peter A, Havenar-Daughton C, Wojcechowskyj JA, Case JB, Chen RE, Kaiser H, Montiel-Ruiz M, Meury M, Czudnochowski N, Spreafico R, Dillen J, Ng C, Sprugasci N, Culap K, Benigni F, Abdelnabi R, Foo SC, Schmid MA, Cameroni E, Riva A, Gabrieli A, Galli M, Pizzuto MS, Neyts J, Diamond MS, Virgin HW, Snell G, Corti D, Fink K, Veesler D. . Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms. Science , 2020 Sep 2024:eabe3354. doi: 2010.1126/science. abe3354. Epub ahead of print. PMID: 32972994. (2020). Abe, K. T. et a\. A simple protein-based surrogate neutralization assay for SARS-CoV-2. JCI Insight 5, doi: 10.1172/jci. insight.142362 (2020). Robbiani, D. F. et al. Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals. bioRxiv, doi: 10.1101/2020.05.13.092619 (2020). Qu, J. et al. Profile of IgG and IgM antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clin Infect Dis , doi: 10.1093/cid/ciaa489 (2020). Voss, C. et al. Epitope-specific antibody responses differentiate COVID-19 outcomes and variants of concern. JCI Insight 6, doi: 10.1172/jci. insight.148855

(2021).

Claims

CLAIMS What is claimed is:

1 . A method of detecting presence of SARS-CoV neutralizing immunoglobulins in a sample taken from a subject, the sample having immunoglobulins, comprising: (a) contacting an aliquot of the sample taken from the subject with particles covered with the SARS-CoV receptor binding domain (RBD) of the Spike (S) protein (S-RBD) under conditions that allow the particles covered with the S-RBD to agglutinate in the presence of immunoglobulins against the S-RBD (agglutination conditions) and obtaining a first agglutination score, (b) mixing particles covered with the S-RBD with angiotensin converting enzyme 2

(ACE2) to form a mixture, and

(c) adding the mixture to another aliquot of the sample taken from the subject under the same agglutination conditions as in step (a), and obtaining a second agglutination score, wherein a reduction in the first agglutination score of the sample in Step (a) relative to the second agglutination score of the ACE2 mixture in Step (c) is indicative of the presence of the SARS-CoV neutralizing immunoglobulins in the sample taken from subject.

2. The method of claim 1 , wherein, the S-RBD is the S-RBD of SARS-CoV-2.

3. The method of any one of claims 1 and 2, wherein the method of further comprises comparing the first agglutination score and the second agglutination score with the agglutination scores of control samples having known amounts of neutralizing immunoglobulins, thereby providing a measurement of the neutralizing immunoglobulins in the sample based on said comparison.

4. The method of any one of claims 1 to 3, wherein the particles are red blood cells, latex particles, polystyrene microspheres, or microspheres or particles made of other non-latex and non-polystyrene polymers,

5. The method of any one of claims 1 to 4, wherein the sample includes one or more of blood, serum, plasma, urine, cerebrospinal fluid and saliva and derivatives thereof.

6. A method for detecting a humoral immune response to SARS-CoV in a subject, comprising the steps of: (a) contacting a sample having immunoglobulins taken from said subject with particles covered with a SARS-CoV antigen; and (b) detecting occurrence of agglutination of the particles covered with the SARS-CoV antigen, wherein occurrence of agglutination of the particles covered with the SARS-CoV antigen indicates a positive humoral immune response to SARS-CoV in the subject.

7. The method of claim 6, wherein the SARS-CoV antigen comprises a spike (S) protein of the SARS-CoV, a part of the S protein, a receptor binding domain (RBD) of the S protein, an epitope of the S protein, a nucleocapsid (N) protein of SARS-CoV-2, a part of the N protein, an RNA binding domain of the N protein (N-RBD), an epitope of the N protein, a matrix (M) protein (aka E1 membrane glycoprotein) of the SARS- CoV, a part of the M protein, an epitope of the M protein, or any combination thereof.

8. The method of claim 6, wherein the SARS-CoV antigen is a single peptide antigen of a protein, or more than one copy of the same single peptide antigen in tandem, or a combination of two or more different single peptide antigens from the same protein or from a different protein of the SARS-CoV that are either arranged in tandem in a single polypeptide or are mixed together.

9. The method of claim 8, wherein the single peptide antigen is an epitope of the S protein, an epitope of the N protein or an epitope of the M protein.

10. The method of claim 8 or claim 9, wherein the single peptide antigen includes no more than 25 amino acid residues, or no more than 20 amino acid residues, or no more than 15 amino acid residues, or no more than 10 amino acid residues.

11. The method of claim 9, wherein the epitope of the S protein, the epitope of the N protein or the epitope of the M protein is selected from the epitopes of T able 3.

12. The method of claim 6, wherein the antigen is an epitope selected from the group consisting of SEQ ID NOs: 48, 82, 86 and 97.

13. The method of claim 6, wherein the antigen is an epitope is selected from the group consisting of SEQ ID NOs: 116 and 139.

14. The method of claim 6, wherein the antigen is an epitope of a variant of concern (VOC epitope) of the SARS-CoV to detect the VOC specific immunoglobulins.

15. The method of claim 14, wherein the VOC epitope is selected from the group consisting of SEQ ID NOs: 150-220 and 221-227.

16. The method of claim 6, wherein the particles include particles covered with two or more different SARS-CoV antigens.

17. The method of claim 16, wherein the particles include a population of particles covered with the S protein or an epitope of the S protein, a population of particles covered with N protein or an epitope of the N protein, and a population of particles covered with both the S and N protein or multiple epitopes from the S and N proteins.

18. The method of any one of claims 6 to 17, wherein the particles are red blood cells or latex particles.

19. The method of any one of claims 6 to 18, wherein the sample includes one or more of blood, serum, plasma, urine, cerebrospinal fluid and saliva and derivatives thereof.

20. The method of any one of claims 6 to 19, wherein the SARS-CoV is SARS-CoV- 2.

21. A method of measuring the level of immunoglobulins against a SARS-CoV in a sample from a subject that contains immunoglobulins, comprising the steps of: (a) contacting the sample with particles covered with a SARS-CoV antigen under conditions that allow the particles covered with the SARS-CoV antigen to agglutinate in the presence of immunoglobulins against SARS-CoV; and

(b) detecting occurrence or absence of particle agglutination in the sample, said occurrence or absence of particle agglutination in the sample having an agglutination area, and correlating the agglutination area of the sample with the agglutination areas of multiple control agglutinations, each control agglutination area containing known concentrations of immunoglobulins against SARS-CoV, to provide a measure of the level of the immunoglobulins against the SARS-CoV in the sample.

22. The method of claim 21 , wherein the SARS-CoV antigen comprises a spike (S) protein of SARS-CoV, a part of the S protein, a receptor binding domain (RBD) of the S protein, an epitope of the S protein, a nucleocapsid (N) protein of SARS-CoV, a part of the N protein, an RNA binding domain of the N protein (N-RBD), an epitope of the N protein, a matrix (M) protein (aka E1 membrane glycoprotein) of SARS-CoV, a part of the M protein, an epitope of the M protein, or any combination thereof.

23. The method of claim 21 , wherein the SARS-CoV antigen is a single peptide antigen of a protein, or more than one copy of the same single peptide antigen in tandem, or a combination of different single peptide antigens from the same protein or from different proteins of SARS-CoV that are either arranged in tandem in a single polypeptide or are mixed together.

24. The method of claim 23, wherein the single peptide antigen is an epitope of the S protein, an epitope of the N protein or an epitope of the M protein.

25. The method of claim 22 or claim 23, wherein the single peptide antigen includes no more than 25 amino acid residues, or no more than 20 amino acid residues, or no more than 15 amino acid residues, or no more than 10 amino acid residues.

26. The method of claim 23, wherein the epitope of the S protein, the epitope of the N protein or the epitope of the M protein is selected from the epitopes of T able 3.

27. The method of any one of claims 21 to 26, wherein the particles include particles covered with different SARS-CoV antigens.

28. The method of claim 27, wherein the particles include a population of particles covered with the S protein, a part of the S protein, or an epitope of the S protein, a population of particles covered with the N protein, a part of the N protein or an epitope of the N protein, and a population of particles covered with both the S and N protein or multiple epitopes from the S and N proteins.

29. The method of claim 21 , wherein the antigen is an epitope selected from the group consisting of SEQ ID NOs: 82 and 97.

30. The method of claim 21 wherein the antigen is an epitope is selected from the group consisting of SEQ ID NOs: 116 and 139.

31. The method of claim 21 , wherein the antigen is an epitope of a variant of concern (VOC epitope) of the SARS-CoV and the measure provides the level of VOC specific immunoglobulins against the SARS-CoV in the sample.

32. The method of claim 31 , wherein the VOC epitope is selected from the group consisting of SEQ ID NOs: 150-220 and 221-227.

33. The method of any one of claims 21 to 32, wherein the particles are red blood cells, latex particles, polystyrene microspheres, or microspheres or particles made of other non-latex and non-polystyrene polymers.

34. The method of any one of claims 21 to 33, wherein the sample includes one or more of blood, serum, plasma, urine, cerebrospinal fluid and saliva and derivatives thereof.

35. The method of any one of claims 21 to 34, wherein each control agglutination containing the known concentration of the immunoglobulins against SARS-CoV is assigned a score between 0 and 4 based on the agglutination intensity, 0 corresponds to no agglutination, 1 corresponds to about 25% agglutination, 2 corresponds to about 50% agglutination, 3 corresponds to about 75% agglutination, and 4 corresponds to about 100% agglutination, and wherein step (c) further comprises assigning a score to the sample based on the comparison to the agglutination intensity of each control agglutination.

36. The method of any one of claims 21 to 35, wherein the SARS-CoV is SARS-CoV- 2.

37. A method for semi-quantitatively measure of a titer of antibody against a SARS- CoV in a subject, comprising the steps of:

(a) contacting a sample containing immunoglobulins from said subject with particles covered with a SARS-CoV antigen;

(b) allowing the particles covered with the SARS-CoV-2 antigen to agglutinate into a clump area;

(c) calculating the percentage of agglutination based on agglutination/clumps area relative to the total particle reaction area, and

(d) plotting the percentage of agglutination against an antibody titer curve to obtain the titer of antibody against the SARS-CoV in the subject.

38. The method of claim 37, wherein the SARS-CoV antigen comprises a spike (S) protein of the SARS-CoV, a part of the S protein, a receptor binding domain (RBD) of the S protein, an epitope of the S protein, a nucleocapsid (N) protein of SARS-CoV, a part of the N protein, an RNA binding domain of the N protein (N-RBD), an epitope of the N protein, a matrix (M) protein of the SARS-CoV, a part of the M protein, an epitope of the M protein (aka E1 membrane glycoprotein), or any combination thereof.

39. The method of claim 37, wherein the SARS-CoV antigen is a single peptide antigen of a protein, or more than one copy of the same single peptide antigen in tandem, or a combination of different single peptide antigens from the same protein or from different proteins of the SARS-CoV that are either arranged in tandem in a single polypeptide or are mixed together.

40. The method of claim 39, wherein the single peptide antigen is an epitope of the S protein, an epitope of the N protein or an epitope of the M protein.

41. The method of claim 39 or claim 40, wherein the single peptide antigen includes no more than 25 amino acid residues, or no more than 20 amino acid residues, or no more than 15 amino acid residues, or no more than 10 amino acid residues.

42. The method of claim 40, wherein the epitope of the S protein, the epitope of the N protein or the epitope of the M protein is selected from the epitopes of T able 3.

43. The method of any one of claims 37 to 42, wherein the particles include particles covered with different SARS-CoV antigens.

44. The method of claim 37, wherein the particles include a population of particles covered with the S protein, a part of the S protein or an epitope of the S protein, and a population of particles covered with the N protein, a part of the N protein or an epitope of the N protein, and a population of particles covered with both the S and N protein or multiple epitopes from the S and N proteins.

45. The method of claim 37, wherein the antigen is an epitope selected from the group consisting of SEQ ID NOs: 82 and 97.

46. The method of claim 37, wherein the antigen is an epitope is selected from the group consisting of SEQ ID NOs: 116 and 139.

47. The method of claim 37, wherein the antigen is an epitope of a variant of concern (VOC epitope) of the SARS-CoV.

48. The method of claim 47, wherein the VOC epitope is selected from the group consisting of SEQ ID NOs: 150-220 and 221-227.

49. The method of any one of claims 37 to 48, wherein the particles are red blood cells, latex particles, polystyrene microspheres, or particles made of other non-latex polymers and polystyrene polymers.

50. The method of any one of claims 37 to 49, wherein the sample includes one or more of blood, serum, plasma, urine, cerebrospinal fluid and saliva and derivatives thereof.

51. The method of any one of claims 37 to 50, wherein the SARS-CoV is SARS-CoV- 2.

52. A method for determining coronavirus disease 2019 (COVID-19) severity comprising:

(a) collecting a sample from a subject whose COVID-19 severity needs to be determined, (b) mixing the sample with particles coated with a SARS-CoV-2 epitope that binds to a SARS-CoV-2 immunoglobulin under conditions that promote agglutination of the particles coated with the SARS-CoV-2 epitope with SARS-CoV-2 immunoglobulin present in the sample (agglutination conditions), and measuring a degree of agglutination, (c) comparing the degree of agglutination obtained in step (b) with (i) a negative control degree of agglutination obtained by mixing a sample from an individual that is SARS- CoV-2 negative with the particles coated with a SARS-CoV-2 epitope that binds to a SARS-CoV-2 immunoglobulin underthe same agglutination conditions, (ii) a moderate control degree of agglutination obtained by mixing a sample from an individual with known moderate COVID-19 symptoms with the particles coated with a SARS-CoV-2 epitope that binds to a SARS-CoV-2 immunoglobulin under the same agglutination conditions, and (iii) a severe control degree of agglutination obtained by mixing a sample from an individual with known severe COVID-19 symptoms with the particles coated with a SARS-CoV-2 epitope that binds to a SARS-CoV-2 immunoglobulin under the same agglutination conditions, and

(d) determining COVID-19 severity based on the comparison of step (c).

53. A method for determining coronavirus disease 2019 (COVID-19) outcome comprising:

(a) collecting a sample from a subject whose COVID-19 outcome needs to be determined, (b) mixing the sample with particles coated with a SARS-CoV-2 epitope that binds to a SARS-CoV-2 immunoglobulin under conditions that promote agglutination of the particles coated with the SARS-CoV-2 epitope with SARS-CoV-2 immunoglobulin present in the sample (agglutination conditions), and measuring a degree of agglutination, (c) comparing the degree of agglutination obtained in step (b) with (i) a negative control degree of agglutination obtained by mixing a sample taken from an individual that is SARS-CoV-2 negative with the particles coated with a SARS-CoV-2 epitope that binds to a SARS-CoV-2 immunoglobulin under the same agglutination conditions, (ii) a live control degree of agglutination obtained by mixing a sample taken from an individual with that is COVID-19 positive with the particles coated with a SARS-CoV-2 epitope that binds to a SARS-CoV-2 immunoglobulin under the same agglutination conditions, and (iii) a fatal control degree of agglutination obtained by mixing a sample taken from an individual that died of COVID-19 with the particles coated with a SARS-CoV-2 epitope that binds to a SARS-CoV-2 immunoglobulin under the same agglutination conditions, and

(d) determining COVID-19 outcome based on the comparison of step (c).

54. The method of any one of claims 52 and 53, wherein SARS-CoV-2 epitope is one SARS-CoV-2 epitope or combination of two or more SARS-CoV-2 epitopes selected from the SARS-CoV-2 epitopes of Table 3.

55. The method of claim 54, wherein the one SARS-CoV-2 epitope or combination of two or more SARS-CoV-2 epitopes is selected from the group consisting of SEQ ID NOs: 48, 82, 86 and 116.

56. The method of claim 54, wherein the antigen is an epitope of a variant of concern (VOC epitope) of the SARS-CoV and the measure provides the level of VOC specific immunoglobulins against the SARS-CoV in the sample.

57. The method of claim 56, wherein the VOC epitope is selected from the group consisting of SEQ ID NOs: 150-220 and 221-227.

58. A method of identifying subjects that have been infected with SARS-CoV-2 and subjects that have been vaccinated with a COVID-19 vaccine and have not been infected with SARS-CoV-2, the method comprising:

(a) mixing a sample taken from a subject with particles coated with SARS-CoV-2 nucleocapsid (N) protein under conditions that promote agglutination of the particles coated with the SARS-CoV-2 N protein with anti-N SARS-CoV-2 immunoglobulins,

(b) mixing a sample taken from the same subject with particles coated with SARS- CoV-2 spike (S) protein under conditions that promote agglutination of the particles coated with the SARS-CoV-2 S protein with anti-S SARS-CoV-2 immunoglobulins, and

(c) measuring a level of agglutination of the particles coated with SARS-CoV-2 N protein in (a) and a level of agglutination of the particles coated with SARS-CoV-2 S protein in (b), wherein when the level of agglutination of the particles coated with SARS-CoV-2 N protein are detectable, then the subject has been infected with SARS-CoV-2, and when the level of agglutination of the particles coated with SARS-CoV-2 S protein are detectable, and the level of agglutination of the particles coated with SARS-CoV- 2 N protein are not detectable, then the subject has been vaccinated but not infected with SARS-CoV-2.

59. The method of claim 58, where the S protein is a part of the S protein, a receptor binding domain (RBD) of the S protein, an epitope of the S protein and the N protein is a part of the N protein, a RNA binding domain (RBD) of the N protein, an epitope of the N protein.

60. A system for detecting immunoglobulins against a target antigen (anti-target antigen immunoglobulins), the system comprising: (a) syringe having a tube that is open at a first end, and a plunger that is slidably mounted inside the tube through a second end of the tube,

(b) dyed particles coated with target antigen (“non-agglutinated particles”) such that when the dyed particles are mixed with anti-target antigen immunoglobulins, the dyed particles coated with the target antigen and the anti-target antigen immunoglobulins form agglutinated particles within the tube, and

(c) a polymer gel preloaded in the tube, the polymer gel having a pore size that allows separation of the agglutinated particles from the non-agglutinated particles when the plunger pushes the agglutinated particles towards the first end.

61. The system of claim 60, wherein the tube is graded with marks that provide a semi- quantitative measurement of the detected immunoglobulins against the target antigen.

62. The system of claim 61 , wherein the marks are graded using samples that produce a specific level of aggregation of about 25%, about 50%, about 75% and about 100%.

63. A method to detect immunoglobulins against a target antigen (“anti-target antigen immunoglobulins”) in a sample that contains immunoglobulins taken from a subject comprising:

(a) mixing the sample with dyed particles coated with the target antigen (“non- agglutinated particles”) to form a mixture, such that when the sample contains anti- target antigen immunoglobulins, the dyed particles coated with the target antigen and the anti-target antigen immunoglobulins form agglutinated particles, (b) loading the mixture to one end of a tube pre-loaded with a neutral hydrogel having a pore size for separating the dyed beads coated with the target antigen when agglutinated from dyed beads coated with the target antigen when non-agglutinated, and

(c) pushing the mixture at the one end of the tube loaded with the neutral hydrogel towards a second end of the tube, wherein the agglutinated particles form a band of agglutinated particles, thereby detecting the immunoglobulins against the target antigen.

64. The method of claim 63, wherein the target antigen is an antigen from a pathogen of interest.

65. The method of claim 64, wherein the target antigen is a SARS-CoV antigen selected from a spike (S) protein of the SARS-CoV, a part of the S protein, a receptor binding domain (RBD) of the S protein, an epitope of the S protein, a nucleocapsid (N) protein of SARS-CoV-2, a part of the N protein, an RNA binding domain of the N protein (N-RBD), an epitope of the N protein, a matrix (M) protein (aka E1 membrane glycoprotein) of the SARS-CoV, a part of the M protein, an epitope of the M protein, or any combination thereof.

66. The method of claim 65, wherein the SARS-CoV antigen is a single peptide antigen of a protein, or more than one copy of the same single peptide antigen in tandem, or a combination of two or more different single peptide antigens from the same protein or from a different protein of the SARS-CoV that are either arranged in tandem in a single polypeptide or are mixed together.

67. The method of claim 66, wherein the single peptide antigen is an epitope of the S protein, an epitope of the N protein or an epitope of the M protein.

68. The method of claim 66 or claim 67, wherein the single peptide antigen includes no more than 25 amino acid residues, or no more than 20 amino acid residues, or no more than 15 amino acid residues, or no more than 10 amino acid residues.

69. The method of claim 67, wherein the epitope of the S protein, the epitope of the N protein or the epitope of the M protein is selected from the epitopes of T able 3.

70. The method of any one of claims 63 to 69, wherein the tube is graded with marks that provide a semi-quantitative measurement of the immunoglobulins against the target antigen, and wherein the method further comprises correlating a position of the band within the tube with the marks to provide said semi-quantitative measurement of the immunoglobulins against the target antigen in the sample.

71. The method according to any one of claims 63 to 70, wherein a position of the band of agglutinated particles within the tube provides semi-quantitative measurement of the level of anti-target antigen immunoglobulins in the subject.

72. An isolated SARS-CoV-2 epitope that binds to a SARS-CoV-2 antibody, wherein said SARS-CoV-2 epitope is selected from the group of epitopes listed in Table 3.

73. An isolated SARS-CoV-2 epitope that binds to a SARS-CoV-2 antibody, wherein said SARS-CoV-2 epitope is selected from the group consisting of SEQ ID NOs: 150-220 and 221-227.