WO2023092126A1 - Rapid, point of care detection of neutralizing antibodies against sars-cov-2 - Google Patents

Abstract

Described are rapid, point of care tests to detect circulating neutralizing antibodies against SARS-CoV-2 in a biological sample obtained from patients.

Description

RAPID, POINT OF CARE DETECTION OF NEUTRALIZING ANTIBODIES AGAINST SARS-COV-2

5 CROSS REFERENCE

This application claims priority to U.S. Provisional Patent Application Serial Numbers 63/281,816 filed November 22, 2021 and 63/320,098 filed March 15, 2022, each incorporated by reference herein in their entirety.

10 SEQUENCE LISTING STATEMENT

A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on October 31, 2022 having the file name “22-0483-WO.xml” and is 20kb in size.

15

BACKGROUND OF THE INVENTION

Coronavirus disease 2019 (COVID-19) is a worldwide pandemic caused by Severe Acute Respiratory' Syndrome coronavirus 2 (SARS-CoV-2) infection. Current SARS-CoV-2

20 testing utilizes RT-PCR of respiratory tract samples to detect virus-specific sequences as well as viral antigen testing.^1,2,6 However, PCR and antigen testing cannot identify previously infected individuals who have developed immunity or individuals with protective immunity from vaccination. Recognition of individuals with protective immunity is critically important for monitoring immunity and managing the pandemic. It is additionally important to provide

25 a method of measuring neutralizing antibodies titer in vaccinated individuals since recent data indicates that lower antibody titers are associated with increased susceptibility to breakthrough infection¹². Thus, for public health efforts to fully succeed in controlling the COVID-19 pandemic, it is critical to develop cost-effective, scalable, and widely available methods to rapidly detect SARS-CoV-2 immunity, monitor neutralizing antibody titer,

30 monitor antibody response in vaccinated individuals, and/or confirm vaccination status.

SUMMARY OF THE INVENTION

Provided are rapid, point of care (e.g. bedside, in an outpatient clinic, or at home) tests to detect circulating neutralizing antibodies (nAbs) against SARS-CoV-2 (the virus causing

- 1 - the current COVID 19 pandemic) in a biological sample obtained from patients. The tests comprise lateral flow test strips and methods of use thereof. The lateral flow test strips and methods described herein use a lateral flow' test strip for detecting the presence of neutralizing antibodies (nAbs) to SARS-CoV-2 in a biological sample from a patient,

5 wherein the test strip comprises a sample application region; a control line comprising an immobilized extracellular domain of the human angiotensin II converting enzyme type 2 (ACE2) receptor; and a test line comprising an immobilized human immunoglobulin G (IgG) binding agent; wherein the biological sample is contacted with a SARS-CoV-2 antigen before or after application to the sample application region thus forming a treated sample, wherein

10 the SARS-CoV-2 antigen is conjugated to a first detectable label. In one embodiment, tire control line is located downstream from the sample application region and the test line is located downstream from the control line. In another embodiment, the test line is located downstream from the sample application region and the control line is located downstream from the test line. The strip is configured such that tire presence of nAbs is determined by

15 detecting the first detectable label captured at the test line, and/or the strip is configured such that the titer of nAbs is determined by calculating the ratio of the amount detectable label captured at the test line to the amount of detectable label captured at the control line. In certain aspects, the biological sample can be contacted with SARS-CoV-2 antigen before application to the sample application region. For example, the biological sample can be

20 contacted with the SARS-CoV-2 antigen for a time sufficient to permit binding between the SARS-CoV-2 antigen and any nAbs in the biological sample thus forming a treated sample and the treated sample is then applied to the sample application region. Alteratively, the biological sample can be contacted with the SARS-CoV-2 antigen on the strip itself; for example, the strip can comprise a conjugation region downstream from the sample

25 application region but upstream of the control and test lines, wherein the conjugation region comprises the soluble (or mobilizable) labeled SARS-CoV-2 antigen. As used herein, a “treated sample” is a sample which has been contacted with the labeled SARS-CoV-2 antigen.

The label of the labeled SARS-CoV-2 antigen is preferably a label that is detectable to

30 the naked eye. A “labeled SARS-CoV-2 antigen” specifically includes a SARS-CoV-2 antigen which is conjugated to a detectable label. Such labels include, but are not limited to, noble metal nanoparticles such as gold nanoparticles, including colloidal gold nanoparticles. The SARS-CoV-2 antigen can comprise all or a portion of the spike protein. For example, the SARS-CoV-2 antigen can comprise all or a portion of the receptor binding domain (RED) of

- 2 - the spike protein. In certain aspects, the SARS CoV 2 antigen comprises all or a portion of the RED of the spike protein and does comprise the full spike protein.

The test line comprises an immobilized human IgG binding agent. Examples of IgG binding agents include of protein A, protein G, protein A/G, protein L, protein L/A, protein

5 L/G, protein M, and protein Z, as well as IgG binding fragments thereof; poly-l-lysine; and an anti-IgG antibody. In certain aspects, the test line comprises an immobilized protein A or an IgG binding fragment thereof.

The control line comprises an immobilized receptor or binding agent for the SARS- CoV-2 antigen including, but not limited to, the extracellular domain of the human

10 angiotensin II converting enzyme type 2 (ACE2) receptor.

The invention also includes a diagnostic kit comprising a test strip described herein and further comprising a labeled SARS-CoV-2 antigen. The kit can further comprise a running buffer and optionally, a chase buffer. In additional aspects, the kit comprises a microcapillary tube.

15 The invention further encompasses a method of detecting the presence or titer of neutralizing antibodies (nAbs) to SARS-CoV-2 in a biological sample of a patient, comprising the steps of: contacting the biological sample with a SARS-CoV-2 antigen for a time sufficient for binding of nAbs present in the sample to bind the SARS-CoV-2 antigen thus forming a treated sample, wherein the SARS-CoV-2 antigen is labelled with or

20 conjugated to a first detectable label; wherein the contacting step occurs before or after application to the sample application region; applying the sample or the treated sample to the sample application region of the test strip and the test sample flows from the sample application region to the control line and the test line; and detecting the first detectable label at the test line and optionally detecting the first detectable label at the control line. The

25 presence of the first detectable label at the test line indicates the presence of nAbs and/or die titer of nAbs is determined by calculating the ratio of the amount first detectable label (or in other words, signal) captured at the test line to the amount of first detectable label captured at die control line. In certain aspects, the treated sample flows from the sample application region to the control line and thereafter to the test line.

30 The invention additionally includes a method of increasing a patient’s protective immunity against SARS-CoV-2, wherein the patient has been previously vaccinated against SARS-CoV-2 and/or wherein the patient has previously suffered a SARS-CoV-2 infection, the method comprising: obtaining a biological sample from said patient; contacting the biological sample with a labeled SARS-CoV-2 antigen for a time sufficient for binding of

- 3 - nAbs present in the sample to bind the SARS CoV 2 antigen thus forming a treated sample, wherein the SARS-CoV-2 antigen is conjugated to a first detectable label; wherein the contacting step occurs before or after application to the sample application region; applying the sample or the treated sample to the sample application region of the test strip so as to

5 permit flow of the test sample from the sample application region to the control line and to the test line; detecting the first detectable label at the test line and optionally detecting the first detectable label at the control line; calculating the titer of nAbs is determined by determining the ratio of the amount first detectable label captured at the test line to the amount of first detectable label captured at the control line; and administering a vaccine

10 booster to said patient if the level of nAbs is below a predetermined threshold level. In certain aspects, the treated sample flows from the sample application region to the control line and thereafter to the test line.

In certain aspects of the methods described herein, the biological sample is contacted with the SARS-CoV-2 antigen before application to the sample application region. In other

15 aspects, the sample is contacted with an absorbent pad impregnated with the SARS-CoV-2 antigen before application to the sample control region. In additional aspects, the sample is collected using a microcapillary tube or test swab. The test swab can be made from any appropriate material, including, for example, cotton, plastic, artificial fibers, or a combination thereof.

20

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Membrane-based assay for detecting antibody-mediated SARS-CoV-2 neutralization. (A) Neutralizing antibodies (NAb) can prevent SARS-CoV-2 infection by blocking the binding of the receptor binding domain (RBD) of the viral spike protein to the

25 ACE2 receptor on the host cell. (B) Schematic for membrane-based assay detecting NAbs able to disrupt the binding of recombinant RBD protein to an ACE2 extracellular domain construct immobilized on a cellulose-based test membrane by protein fusion with a cellulose binding domain (CBD). Binding of non-neutralized RBD protein to the membrane- immobilized ACE2 extracellular domain is detected by CR3022 anti-RBD monoclonal

30 antibody followed by chemiluminescence with a horse radish peroxidase (HRP)-conjugated secondary antibody. (C) Recombinant RBD and ACE2-CBD fusion proteins following affinity purification were analyzed by SDS-PAGE and stained with Coomassie blue. Molecular weight standards are indicated in the first lane. (D) Monoclonal neutralizing

- 4 - antibody inhibition of RBD binding to ACE2 in a dot blot assay. Indicated amounts of ACE2 CBD were immobilized to a cellulose membrane, incubated with RBD and indicated amounts of recombinant monoclonal SARS-CoV-2 neutralizing antibody, followed by detection of RBD bound to ACE2 with CR3022 monoclonal antibody followed by HRP-conjugated

5 secondary antibody. (E) Quantification by background-subtracted integrated density of HRP chemiluminescent signal of RBD bound to ACE2 in dot blots, n=3. Error bars represent standard deviation and p-values comparing indicated conditions were calculated with Student’s T-Test. (F) Schematic diagram of monoclonal antibody inhibition of RBD:ACE2 binding in a lateral flow assay. (G) RBD was mixed with indicated amounts of monoclonal

10 NAb in pre-pandemic human plasma, applied to one end of a cellulose test strip spotted with immobilized ACE2-CBD, allowed to run via lateral flow for 15 min, and then detected with CR3022 monoclonal antibody followed by HRP-conjugated secondary antibody. (H) Quantification by background-subtracted integrated density of HRP chemiluminescent signal of RBD bound to ACE2 in lateral flow strips, n = 5. All error bars represent standard

15 deviation, p values comparing indicated conditions were calculated with Student’s t test.

Figure 2. Lateral flow test strip performance in quantifying SARS-CoV-2 neutralizing titer in patient plasma. (A) Schematic of a rapid assay for detecting SARS- CoV-2 NAb titer in a lateral flow format. The colloidal gold-conjugated RBD protein is mixed with a patient sample and applied to one end of a membrane test strip. The infectious

20 line contains immobilized ACE2 extracellular domain, which captures non-neutralized RBD protein, while RBD protein neutralized by NAbs migrates further to the neutralized tine, where it is captured by immobilized protein A binding to the NAbs. The signal at tire neutralized line divided by the signal at the infectious line (N/I ratio) determines NAb titer. (B) Images of lateral flow neutralization test strip results for patient plasma at indicated time

25 points following BNT162b2 vaccination. (C and D) Time course of N/I ratio and pseudoviral neutralization ID50 following vaccination with either (C) BNT162b2 or (D) mRNA-1273.

30 Figure 3. Lateral flow assay performance relative to alternative methodologies for assessing NAb titer. (A) Performance of LFA neutralization test strip for detecting indicated neutralization ID50 titers, as assessed by receiver operating characteristic area under the curve (ROS-AUC) analysis reporting sensitivity and specificity. A total of 127 plasma samples from patients previously infected with SARS-CoV-2, unvaccinated patients

- 5 - without known prior infection, and patients vaccinated with either BNT16262 or mRNA 1273 were assessed. Sensitivity and specificity for detecting indicated neutralization ID50 titers were calculated for N/I ratios from 0 to 0.7 increasing in increments of 0.001, with sensitivity plotted versus false positive rate (100% minus specificity) to generate each ROC

5 curve. AUC and 95% confidence interval for AUC are displayed for each titer. (B) Comparison of diagnostic performance of N/I ratio, loss of binding signal at I line, and ELISA-based anti-spike total IgG. Sensitivity and specificity for detecting indicated neutralization ID50 titers were calculated for N/I ratios as in (A). I line signal integrated density from 21,000 down to 15,000 signal density units decreasing in increments of 20 and

10 anti-spike IgG levels from 0 to 2,430 mg/dL increasing in increments of 5, with sensitivity plotted versus 100% minus specificity to generate each ROC curve. Diagnostic performance of loss of I signal and anti-spike total IgG were then calculated by ROC-AUC analysis as in (A), with p values comparing AUC with N/I ratio calculated using Pearson correlation coefficient testing.

15 Figure 4. Lateral flow assay for at-home assessment of NAb titer. (A) Schematic of a modified lateral flow assay for detecting SARS-CoV-2 NAb titer with an additional control line to assess success of lateral flow. A mixture of colloidal gold-conjugated RBD protein and free colloidal gold is mixed with a patient sample and applied to one end of a membrane test strip. The I and N lines function similarly to Figure 2A, but a third line with

20 immobilized poly-L-lysine captures free colloidal gold particles to indicate successful lateral flow. (B) Images of lateral flow neutralization test strip results for patient whole-blood samples obtained either pre-pandemic, after vaccination with BNT162b2, or after recovery from COVID-19. (C) Proposed workflow for at-home, or point-of-care, qualitative and quantitative assessment of NAb titer.

25 Figure 5. Recombinant protein reagents. Schematic maps of the domains of exemplary recombinant RBD protein, which contain 8xHis, twin Strep-Tag II, twin FLAG affinity purification tags, andALFA and GCN4 purification tags.

DETAILED DESCRIPTION

30 As used herein, the words “a” and “an” are meant to include one or more unless otherwise specified. For example, the term “a control line” means one or more control lines unless otherwise indicated.

- 6 - The terms neutralizing antibody and nAb and NAb are used interchangeably herein.

One obstacle for controlling the SARS-CoV-2 infectious spread, including breakthrough infections, during the COVID- 19 pandemic is the difficulty of identifying

5 individuals with protective immunity. As more people are vaccinated against COVID- 19, there is a need to detect the presence of neutralizing antibodies and/or to monitor neutralizing antibody titer. The ability to identify individuals with protective immunity is vitally important since these individuals can resume activities with fewer restrictions, including returning to school and the workplace. In addition such individuals represent a source of therapeutic

10 convalescent serum. 'There is also a need to monitor neutralizing antibody titer of vaccinated individuals and/or to provide longitudinal monitoring of antibody response after vaccination. Furthermore, there will also be situations where there is a need to quickly confirm vaccination status. Here we describe addressing the needs for identifying individuals with protective immunity by producing scalable, bedside point-of-care blood tests to detect

15 neutralizing antibodies and/or to measure neutralizing antibody titer. The test strip and method described herein can also be used to provide a measurement of antibody titer and/or detect the presence of neutralizing antibodies in a biological sample obtained from a subject Such biological samples include, but are not limited to, blood, serum, saliva, and abrasive gum swab. The methods described herein can be used to confirm vaccination status (whether

20 or not an individual has been vaccinated against CO VID- 19) and/or to monitor titer of neutralizing antibodies after vaccination or after COVID- 19 infection at one or more time points, e.g., providing longitudinal monitoring of antibody titer. The lateral flow assay and methods described herein can detect neutralizing antibodies using a small volume of biological sample for example, using one drop of patient blood or gum or cheek rub, and has

25 a read-out similar to home pregnancy tests. The assays can also measure neutralizing antibody in other sample types including, but not limited to, saliva and gum swab.

In the present invention, the titer of nAb in a sample is directly proportional to the signal at the test line and/or the ratio of signal at the test line to the signal at the control line. In certain aspects, the sample is collected using a microcapillary tube thus providing a

30 consistent volume of sample to the strip.

According to the present invention, the biological sample obtained from the subject is contacted with a SARS-CoV-2 antigen that is labeled with or conjugated to a first detectable label. The SARS-CoV-2 antigen can be further bound to a protein tag or a purification tag. The contact between the biological sample and the SARS-CoV-2 antigen can take place on

- 7 - the test strip; for example, the labeled SARS CoV 2 antigen can be added to the sample while on the test strip and/or be present at a conjugation region or conjugation paid of the test strip which region is upstream from the test and/or control lines. This contact can alternatively or additionally take place before the sample is applied to the test strip. For example, the

5 biological sample can be contacted with the labeled SARS-CoV-2 antigen thus forming a treated sample, and the treated sample is then applied to the test strip (e.g., at the sample application region). In certain aspects, the biological sample is contacted with an absorbent pad impregnated with the SARS-CoV-2 antigen. In further aspects, the absorbent pad which has been contacted with the biological sample can then be contacted with a running buffer to

10 prepare a treated sample which is then applied to the test strip. When the biological sample is contacted with the labeled SARS-CoV-2 antigen before application to the test strip, the test strip need not comprise a conjugation region.

The sample can be obtained from an animal subject. The animal subject can, for example, be a mammalian subject. Preferably the animal subject is a human subject. The

15 subject can be an individual who has been diagnosed or was previously diagnosed with COVID-19 (e.g., tested positive for COVID-19 using a RT-PCRtest or an antigen test). The subject can also be an individual that has never been diagnosed with COVID-19. The subject can be an individual that has been vaccinated against SARS-CoV-2 infection or COVID-19. The subject can be an individual suffering from “long COVID” (for example, individuals that

20 were infected with SARS-CoV-2 but continue to experience symptoms after recovering from die initial stage of illness). The subject can also be an individual w^rhose vaccination status is unknown. “COVID-19” and “SARS-CoV-2 infection” can be used interchangeably herein.

The SARS-CoV-2 antigen can be any portion of a SARS-CoV-2 viral protein that binds to neutralizing antibodies and to an immobilized binding partner or receptor of the

25 SARS-CoV-2 antigen at the test line. For example, the SARS-CoV-2 antigen can be any portion or fragment a SARS-CoV-2 viral protein that binds the extracellular domain of the ACE2 receptor. In certain aspects, the SARS-CoV-2 antigen is a spike protein or a fragment thereof, such as a fragment comprising the receptor binding domain (RED). ACE2 is the human cell surface protein that serves as the receptor to which SARS-CoV-2 RED binds to

30 initiate infection.

Neutralizing antibodies present in the biological sample will bind to the SARS-CoV-2 antigen (for example, soluble RED fragments or soluble fragments that comprise RED), since in tiie body, true neutralizing antibodies will bind to the SARS-CoV-2 antigen and disrupt the virus’s ability to attach to and infect human cells. The diffusion of the sample across the

- 8 - lateral flow membrane will then cany both unbound and any antibody bound labeled SARS CoV-2 antigen across the membrane by lateral flow, and then reach the control line. The control line can, for example, contain or comprise an immobilized receptor (e.g., a human receptor) or a fragment thereof, or binding partner of SARS-CoV-2 antigen. A receptor for

5 SARS-CoV-2 includes, for example, the ACE2 receptor or a fragment thereof. The immobilized ACE2 receptor fragment can, for example, comprise an extracellular domain of a human cell receptor of SARS-CoV-2, preferably the extracellular domain of a human ACE2 receptor. The ACE2 receptor or fragment thereof, such as the extracellular domain of the ACE2 protein, can be part of a fusion protein or conjugate comprising a moiety that binds to

10 the test strip. For example, the fusion protein or conjugate can comprise the ACE2 receptor or fragment thereof and a cellulose binding domain that binds to the strip comprising cellulose. Engineered or modified ACE2 receptors or fragments thereof can also be used at the test line so long as they are capable of binding to the SARS-CoV-2 antigen. Thus, for example, the amino acid sequence of the ACE2 receptor can be modified and/or post-translationally

15 modified and/or ACE2 receptor can be conjugated with another protein, peptide, or fragment. The ACE2 receptor can, for example, be modified such that it has higher affinity for RED than wild-type ACE2. Non-limiting examples of such engineered ACE2 receptor include, for example, those described in Glasgow et al. (2020), Engineered ACE2 receptor traps potently neutralize SARS-CoV-2, PNAS 117 (45) 28046-28055 and Higuchi et al. (2020), High

20 affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters, biorxiv.org/content/10.1101/2020.09.16.29989 lv2.article-info; the contents of each which are expressly incorporated by reference herein. ACE2 receptors and fragments thereof, modified and engineered versions thereof, including, extracellular domains of ACE2 receptors can be collectively referred to herein as “ACE2 receptor”.

25 Labeled SARS-CoV-2 antigen that is not bound by neutralizing antibodies present in the sample will bind to the immobilized ACE2 receptor on the control line, causing a buildup of the label at the control line. If neutralizing antibodies are bound to the SARS-CoV-2 antigen, the labeled SARS-CoV-2 antigen will not bind to the immobilized ACE2 receptor and will continue diffusing past the control line to the test line. The test line comprises an

30 immobilized IgG binding agent that binds any IgG in the sample. Any nAb present in the sample will be bound to labelled SARS-CoV-2 antigen and that first detectable label will be detectable or measurable at the test line. The amount of nAb is proportional to the amount of first detectable label at the test line. In other words, the greater the amount of nAbs in the sample, the more labelled SARS-CoV-2 antigen will be bound to the test line and the less

- 9 - labelled SARS CoV 2 antigen will be bound at the control line. The level of signal at the control and/or test line can be quantified visually (e.g., with the naked eye) or with an optical reader or a handheld computing device (such as a smartphone camera). The signal intensity at the test line is directly correlated with neutralizing antibody titer. In other aspects, the ratio

5 of signal intensity at the test line to that at the control line can be used to provide a measure of nAb titer (as discussed in more detail below). As used herein, the presence of nAbs is “directly proportional” to the signal intensity when, for example, there is an absence of signal at the test line or there is a decreased signal at the test line (decreased, for example, as compared to that of a control sample comprising no nAbs) and this indicates a lack or a low

10 level of nAbs in the biological sample. The assay and methods described herein can additionally comprise applying a control sample to the test strip, wherein the control sample is a biological sample comprising no nAbs or containing a known amount/concentration of nAbs and measuring the signal intensity of the control sample at the test line and optionally, die control line. The optical reader or computing device can compute the intensity of the test

15 line. Alternatively or additionally, the signal can be detected by the naked eye; for example, when a high titer of nAbs are present in the sample, the signal at the test line will be absent or will be of high intensity.

The test strip can alternatively be configured such that the test line is upstream from the control line such that a sample applied at the sample application region will first flow to

20 the test line and then to the control line thereafter. Neutralizing antibodies bound to the labelled SARS-CoV-2 antigen bind at the test line. Then any labeled SARS-CoV-2 antigen that is not bound by neutralizing antibodies present in the sample will diffuse past the test line and bind to the immobilized ACE2 receptor at the control line, causing a build-up of the label at the control line.

25 Non-limiting examples of labels that can be used (e.g., for the first detectable label, the second detectable label or any other label used in the assay) include, but are not limited to, a colloid gold nanoparticle, a colored latex bead, a colored microparticle or nanoparticle, a magnetic particle, a carbon nanoparticle, a selenium nanoparticle, a silver nanoparticle, a fluorescent particle or nanoparticle, and a quantum dot. The label can also be a noble metal

30 nanoparticle. The label can be selected from metallic particles such as gold or silver particles, or polymeric particles such as latex beads, and polystyrene particles, wherein the particles encapsulate visible or fluorescent dyes. In another aspect, the label is an enzymatic label such as horseradish peroxidase. In certain aspects, the label is visible to the naked eye. In additional aspects, the first detectable label is a gold nanoparticle. The strip can additionally

- 10 - comprise a second detectable label, for example, bound to a control protein. The second detectable label can be the same or different from the first detectable label.

The SARS-CoV-2 antigen can, for example, comprise the membrane protein or a fragment thereof, the spike protein or a fragment thereof, tire envelope protein or a fragment

5 thereof, or the nucleoprotein or a fragment thereof. In certain aspects, the SARS-CoV-2 antigen comprises all or a portion of the spike protein. In yet additional aspects, the SARS- CoV-2 antigen comprises all or a portion of the receptor binding domain (RBD) of the spike protein. The SARS-CoV-2 antigen can, for example, be recombinant or recombinantly produced. The SARS-CoV-2 antigen can be a portion of an infectious agent that infects

10 mammals, and preferably infects humans.

The control line can comprise an immobilized receptor or binding partner of the SARS-CoV-2 antigen, such as the immobilized ACE2 protein or a fragment thereof such as the extracellular domain of an ACE2 receptor or a fragment thereof. The ACE2 protein or fiagment thereof can be recombinant or recombinantly produced. In certain aspects, the lS immobilized ACE2 protein comprises the extracellular domain of an ACE2 receptor and optionally is recombinant or recombinantly produced. The extracellular domain of the ACE2 receptor can comprise all or a fiagment of the extracellular domain of the ACE2 receptor so long as it is capable of binding to the SARS-CoV-2 antigen. The extracellular domain of an ACE2 receptor can be the extracellular domain of human ACE2 receptor; for example, a

20 recombinantly produced human ACE2 receptor. In other embodiments, the extracellular domain of an ACE2 receptor can be the extracellular domain of a mammalian ACE2 receptor; for example, a recombinantly produced mammalian ACE2 receptor. The ACE2 receptor or fiagment thereof can, for example, be immobilized at the control line by covalent coupling and/or affinity binding. For example, the ACE2 receptor can be biotinylated and

25 can bind to the test line by biotin:streptavidin binding. In another example, the ACE2 receptor can be coupled to a cellulose binding domain, e.g., forming a fusion protein (and the strip at the control line comprises cellulose).

The test line comprises an immobilized human IgG binding agent. Examples of IgG binding agents include protein A, protein G, protein A/G, protein L, protein L/A, protein L/G,

30 protein M, and protein Z, as well as IgG binding fragments thereof; poly-l-lysine; and an anti- IgG antibody. In certain aspects, the test line comprises an immobilized protein A or an IgG binding fiagment thereof. Additional examples of human IgG binding agents are described, for example, in Sidorin et al. (2011), Biochemistry (Moscow) 76(3): 295-308; Biotz et al. (2020), Frontiers in Microbiology 11: 10.3389/finicb.2020.00685; Choe et al. (2016),

- 11 - Materials 9, 994: doi: 10.3390/ma9120994; the contents of each of which are expressly incorporated by reference herein. The immobilized IgG binding agent can also be a fusion protein, for example, a fusion protein comprising protein A, protein G, protein A/G, protein L, protein L/A, protein L/G, protein M, and protein Z or an IgG binding fragment of any of

5 thereof. Such fusion proteins are described for example in Choe et al. The immobilized IgG binding agent can also be a peptide, such as a short cyclic peptide, that binds to human IgG. Exemplary peptides that bind IgG are described in Choe et al. and include for example, PAM, D-PAM, D-PAM-$, TWKTSRISIF (SEQ ID NO: 1), FGRLVSSIRY (SEQ ID NO:2), Fc-HI, Fc-IH-(Sepharose), FcBP-2, Fc-IIMC, EPIHRSTLTALL (SEQ ID NO:3), APAR, FcRM,

10 HWRGWV(SEQ ID NO:4), HYFKFD (SEQ ID NO:5), HFRRHL (SEQ ID NO:6), HWCitGWV (SEQ ID NO:7), D2AAG (SEQ ID NO:8), DAAG (SEQ ID NO:9), cyclo[(Na- Ac)S(A)-RWHFYK-Lact-E] (SEQ ID NO: 10), cyclo[Na-Ac]-Dap(A)- RWHFYK-Lact-E] (SEQ ID NO: 11), cyclo[Link-M-WFRHYK]m (SEQ ID NO: 12), NKFRGKYK(SEQ ID NO: 13), NARKFYKG(SEQ ID NO: 14), FYWHCLDE(SEQ ID NO: 15), FYCHWALE (SEQ

15 ID NO: 16), FYCHTIDE (SEQ ID NO: 17), Dual 1/3, RRGW (SEQ ID NO: 18), and KHRFNKD (SEQ ID NO: 19).

The biological sample can be any sample which may contain neutralizing antibodies. For example, the biological sample can be a blood sample, a serum sample, saliva sample, or an abrasive gum swab sample.

20 The sample application region (a region of the test strip to which the sample or treated sample is applied) is upstream of the control line or region and upstream of the test line. The sample application region can additionally provide pH control/modification and/or specific gravity control/modification of the sample applied, and/or removal or alteration of components of the sample which may interfere or cause non-specific binding in the assay,

25 and/or direct and control sample flow to the test region. The terms “control line” and “control region” are used interchangeably herein. Similarly, the terms “test line” and “test control region” are used interchangeably herein.

When present, the conjugation region or conjugation pad is upstream of the control and test lines and downstream of the sample application region. The conjugation region can

30 comprise the labeled RED (or a labeled fragment comprising the RBD or labeled SARS- CoV-2 antigen) and/or the labeled anti-control antibody in soluble or mobilizable form. Illustrative materials material for the conjugation region (e.g., conjugation pad) include, but are not limited to, cellulose, nitrocellulose, fiberglass, cotton, woven or nonwoven paper etc.

- 12 - The terms conjugation region and conjugate region and conjugation pad are used interchangeably herein.

The test strips described herein can further comprise an absorbent region or pad at the distal end of the test strip that collects the processed sample and/or can cause the sample to

5 move from the sample application region toward the absorbent region or pad. The test strip can also comprise a solid support which provides support for the pads and membranes of the lateral flow test strip. A buffer can be used to dilute and/or pre-treat the sample. The buffer can comprise, a salt, a mild detergent/surfactant, and an agent that inhibits or prevents nonspecific binding. Exemplary buffers can, for example, comprise phosphate buffered saline

10 (PBS) and/or saline or NaCl and non-ionic surfactants. In additional examples, buffers can comprise Bronidox, for example 0.1% (w/v) Bronidox. Exemplary buffers are shown below in Table 1.

Table 1

Buffer Components in addition to 0.1% (w/v) Bronidox;

3H = IX Diluent-3H [10X = 25% X305 Z15% sue / 5% C-BL / 10X

TEJ + 0.9% NaCl

3H-11 3% Triton X305 / 5% C-BL / 1% NaCl / IX TE

3H-10 5% X305 / 5% C-BL / 0.9% NaCl / IX TE

3H-9 5% X305 / 2% sucrose (sue) / 5% C-BL / 0.5% NaCl / IX TE

15 The assay can additionally comprise a blood separation pad and/or a method to separate red blood cells and other cells or solid components from the sample. For example, a blood separation pad can be used. In certain additional aspects, the strip comprises an

- 13 - absorbent pad impregnated with the labelled SARS CoV 2 antigen wherein the sample application region is located within or downstream of the absorbent pad.

The membrane used in the lateral flow assay strip of the present invention can be made of a variety of materials which the sample to be tested can pass or move through and

5 that are known for a person skilled in the art. For example, the materials used to form the membrane can include, but are not limited to, natural, synthetic, or naturally occurring materials that are synthetically modified, such as polysaccharides (for example, cellulose materials such as paper and cellulose derivatives as cellulose acetate and nitrocellulose); polyether sulfone; nylon; silica; inorganic materials, such as deactivated alumina,

10 diatomaceous earth, MgSOt, or other inorganic finely divided material uniformly dispersed in a porous polymer matrix such as vinyl chloride, vinyl chloride-propylene copolymer, and vinyl chloride-vinyl acetate copolymer; cloth, both naturally occurring (for example, cotton) and synthetic (for example, rayon); porous gels, such as silica gel, agarose, dextran, and gelatin; polymeric films, such as polyacrylamide; and the like. In a preferred embodiment, the

15 membrane comprises nitrocellulose or cellulose. In certain embodiments, the strip comprises is paper. In some embodiments, the strip comprises cellulose, such as filter paper, chromatographic paper, nitrocellulose, and cellulose acetate.

In certain embodiments, the method comprises contacting the biological sample with a SARS-CoV-2 antigen for a time sufficient for binding of nAbs present in the sample to

20 bind the SARS-CoV-2 antigen thus forming a treated sample, wherein the SARS-CoV-2 antigen is labeled with a first detectable label and wherein the contacting step occurs before application to the sample application region. The treated sample can further comprise a buffer, for example, a running buffer. In certain aspects, the biological sample is contacted with an absorbent pad impregnated with the labelled SARS-CoV-2 antigen and optionally,

25 the treated pad is combined with a buffer (e.g., the running buffer) to form the treated sample. The treated sample is applied to the sample application region and flows from the sample application region to the control and test lines in an order that depends on the configuration of die test strip. For example, the strip can be configured such that the control line is downstream of the sample application region and the test line is downstream of the control

30 tine thus the treated sample applied to the sample application region flows to the control line and thereafter to the test line. When the method entails contacting the biological sample with a SARS-CoV-2 antigen prior to application to the sample application region, the test strip may or may not comprise a conjugation region. Once the sample has migrated past the test line and the control line, the first detectable label is detected at the test line and optionally the

- 14 - first detectable label is detected at the control line. The amount of first detectable label at the test line is proportional to the titer of nAbs in the sample. In certain aspects, the method comprises measuring the amount of label at the test line and measuring the amount of label at the control line, wherein the ratio of the amount of first detectable label at the test line to the

5 amount of first detectable label at the control line provides a measure of the titer of nAbs in the sample. The amount of detectable label at the test line or the control line can be detected with a handheld computing device including, but not limited to, a smartphone camera. In other aspects, the presence or absence of the first detectable label at the test line and optionally the presence of the first detectable label at the control line is determined visually,

10 e.g., with the naked eye. As discussed above, the absence or low amount of first detectable label at the test line is indicative of the lack of or low titer of nAbs in the sample.

In specific embodiments, the invention encompasses a rapid point-of-care lateral flow assay that quantifies neutralizing antibody titers against SARS-CoV-2 using a biological sample, such as human blood, saliva, or a gingival mucosa oral swab. This test is based on

15 brief incubation of the human test sample with recombinant SARS-CoV-2 spike protein receptor binding domain (RBD) that has been conjugated to colloidal gold. This brief incubation allows any neutralizing antibody in the sample to bind to RBD and neutralize its ability to bind to the human cell surface receptor, the ACE2 protein. The mixture is then run on a lateral flow assay membrane described herein where recombinant extracellular domain

20 of ACE2 has been immobilized to a control line. If neutralizing antibody is present, then less gold-conjugated RBD will be captured at the control line via binding to the immobilized ACE2 as compared to that at the test line. The binding of gold-conjugated RBD at the test and/or control lines creates a colloidal gold signal at the test line and/or control. If neutralizing antibody is present in the sample, the gold-conjugated RBD will be blocked from

25 binding to the immobilized ACE2 extracellular domain, decreasing the signal at the control line and increasing the signal at the test line. The signal at the test line and/or the relative levels of signal at the control and test lines can be quantified visually, or with an optical reader, for example, a simple optical reader such as a smartphone camera. Thus, the signal intensity of the test line is directly correlated with neutralizing antibody titer. Variations in

30 the volume or amount of sample will alter the total amount of neutralizing antibody that is loaded into the test. Thus, in certain aspects, a microcapillary tube can be used to obtain a precise or constant volume of biological sample. In other aspects, an internal control protein can be used, for example, an abundant plasma protein. One such plasma protein is human albumin, which is present in human blood and gingival swab samples and can be used as an

- 15 - internal control. Other plasma proteins that can be used as an internal control include, for example, Factor V and Factor IX. In the test, in addition to the presence of gold-conjugated RBD in the brief incubation step a gold-conjugated monoclonal antibody against the control plasma protein can also be included.

5 In an additional specific embodiment, the gold-conjugated RBD or other SARS-CoV- 2 antigen are located at the conjugation region and are contacted with the biological sample when the sample flows from the sample application through the conjugate region. After the conjugation region, the sample flows to the control and test lines. If no neutralizing antibody is present, the gold-conjugated RBD will be captured at the test line via binding to the

10 immobilized ACE2, thus creating a colloidal gold signal at the test line. If neutralizing antibody is present in the sample, the gold-conjugated RBD will be blocked from binding to the immobilized ACE2 extracellular domain, and bind to immobilized IgG binding agent thus providing a signal at the test line. This signal at the test line and optionally at the control line can be quantified visually, or with an optical reader such as a smartphone camera. Again, the

15 signal intensity of the test line is directly correlated with neutralizing antibody titer.

The tests described herein can provide a signal or test results rapidly, for example, in less than about 60 minutes, preferably less than about 30 minutes, preferably in less than about 20 minutes and preferably in less than about 10 minutes, or less than 5 minutes. The test provides test results with an accuracy of about 90% or greater, preferably about 95% or

20 greater, preferably about 99% or greater and preferably about 100%. In certain aspects, the test strip provides a sensitivity of at least about 80%, or at least about 85%, or at least about 90%, or at least about 92%, or at least about 95%, or at least about 97%. In further aspects, the test strip provides a specificity of at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 92%, or at least about 95%, or at least

25 about 97%. The test strip can be utilized as the point-of-care. A point-of-care test is a test performed at or near the place where the sample is collected and can provide rapid results, e.g., within minutes. Such point-of-care tests can be used at the patient’s bedside, at a physician’s office, in an urgent care setting, at a pharmacy, at a school or university health clinic, at a long term care facility, at airports and other points of entry, and/or at other

30 locations.

The test strip can optionally comprise one or more additional control test lines (e.g., a ‘second control line”, wherein a binding agent for a control protein and/or a protein tag is immobilized. The term “control protein” and “protein control” are used interchangeably herein. The control protein can, for example, be a protein naturally present in the biological

- 16 - sample (e.g., a plasma protein), or can be a protein tag. Non limiting examples of plasma proteins are albumin, Factor V, and Factor IX. The control protein can be detected using antibody sandwich methods that are familiar to those of skill in the art. For example, when the control protein is a plasma protein, the additional or second control line can comprise an

5 immobilized antibody with antigenic specific for the plasma protein (also referred to herein as “an anti-plasma protein antibody’' and the like). As used herein, the term “antibody” includes polyclonal and monoclonal antibodies, full-length antibodies or full length immunoglobulins, as well as antigen-binding fragments thereof, including, but not limited to, Fab, Fv and F(ab')2, Fab', and the like. In certain aspects, an anti-plasma protein antibody

10 immobilized at a second or additional control line is referred to herein as the “first” anti- plasma protein antibody or tire “first” anti-albumin antibody (when tire control protein is albumin). The first anti-plasma protein antibody can be a monoclonal antibody or a polyclonal antibody. In certain aspects, the first anti-plasma protein antibody or anti-albumin antibody is a polyclonal antibody. The biological sample can be contacted with a second

15 antibody (or any other agent) with antigenic specificity for the plasma protein (either before or after application to the sample application region). The second antibody with antigenic specificity for the control protein or plasma protein (or albumin) can be contacted with the biological sample prior to application to the test strip, for example it can be part of the treated sample. Alternatively or additionally, the second antibody can be present at the conjugation

20 region and can bind to the plasma protein or albumin in the sample as it passes through the conjugation region. The second anti-plasma protein antibody can be monoclonal or polyclonal. In certain aspects, the second anti-plasma protein antibody or second antialbumin antibody is a monoclonal antibody. The second antibody with antigenic specificity for the plasma protein can be labeled and as such, this label can then be detected at the

25 control line. This label can be referred to herein as the “second detectable label.” The second detectable label can for, example, be a colored nanoparticle including but not limited to colloidal gold nanoparticles. As will be understood, the strip can comprise more than one additional control lines that detect more than one control protein. For example, the strip can comprise a control line for detecting albumin and a control line for detecting another control

30 protein and/or protein tag in the sample. When the control protein is a plasma protein, for example, albumin, Factor V, or Factor IX, the control protein is present in the present at a relatively constant concentration. Thus, the signal at this control line will not vary depending on the presence or absence of neutralizing antibody but will vary depending on volume of sample applied to the strip, thus serving as a control for test sample amount. When such a test

- 17 - is run, the ratio of signal at the test line to signal at the control line will be inversely proportional to neutralizing antibody titer. The protein/purification tag can, for example, be FLAG, GCN4 and/or ALFA, as described in more detail below.

In one embodiment, the one or more additional control test lines may comprise a

5 control line comprising, for example, poly-L-lysine. This embodiment is particularly useful to capture any free colloidal gold to serve as the run control line.

In certain aspects, the methods described herein are used to identify the presence of nAbs in the biological sample. For example, the test can also provide test results with an accuracy of about 90% or greater, preferably about 95% or greater, preferably about 99% or

10 greater and preferably about 100%.

As described above, the ratio of the signal at the test line to that at the control line (the T/C ratio) can provide a measure of neutralizing antibody titer. In certain aspects, the methods described herein are used to determine neutralizing antibody (nAb) titer. For example, the methods can be used to differentiate samples where the nAb titer in the lower

15 range (e.g., the least protective against breakthrough infection), an intermediate range (e.g., more effective than tire lower range and/or protective against severe disease), or a high range (e.g., more protective than the intermediate range). Since the T/C ratio is directly proportional to nAb titer, a T/C ratio within a predetermined low range corresponds to a low level or range of nAb titer and a T/C ratio within a predetermined high range corresponds to a high level or

20 range of nAb titer. In certain aspects, a low range or level of nAb is less than about 1 : 100, an intermediate range or level is between about 1 : 100 and 1 : 1000, and the high range or level is greater than 1 : 1000 as determined by SARS-CoV-2 pseudovirus neutralization assay in serum samples (Dieterie et al. (2020), Cell Host Microbe 28(3): 486-496.e6¹³; the contents of which are expressly incorporated by reference herein). In other examples, the low,

25 intermediate, and high ranges are correlated to percent efficacy as described in Khoury et al. (2021),¹⁴ the contents of which are expressly incorporated herein. Bergwerk et al. have recently shown that lower antibody titers during the peri-infection period (within a week before SARS-CoV-2 was detected and including the day of SARS-CoV-2 infection) are associated with increased susceptibility to breakthrough infection and lower antibody titers

30 were also associated with higher viral loads.¹² Also, Khoury et al.¹⁴ and Earle et al.¹⁵ Vaccine have shown that level of nAbs is highly predictive of immune protection. Thus, the method described herein can assess risk of susceptibility to breakthrough infection. A breakthrough infection is SARS-CoV-2 infection, for example, as confirmed using RT-PCR assay, at least

- 18 - about 11 or more days after full vaccination. Whether full vaccination occurs after the first or second dose depends on the vaccine. For example, for the Pfizer BNT162b2 mRNA vaccine and the Modema mRNA 1273 mRNA vaccines, full vaccination occurs after the second dose. For the Jannsen COVID- 19 vaccine, full vaccination occurs after a single dose. Additional

5 vaccines include, but are not limited to, NVX-CoV2373, rAD26+S-rAd5-S, ChAdOxl nCoV-19, Ad26.COV2.2, and CoronaVac. The methods can also be used to monitor antibody titer after vaccination or after SARS-CoV-2 infection and the method can entail measurement at different time points after vaccination or infection. The methods can additionally be used to identify patients in need of booster vaccination and can comprise

10 administration of a booster dose of vaccine; for example, if the nAb titer is below a predetermined threshold level, a booster vaccine dose is administered to the patient. In certain aspects, the predetermined threshold level is less than an intermediate titer or level of nAbs. In yet further aspects, the predetermined threshold level is less than a low titer or level of nAbs. Depending on the method, the sample can be obtained before vaccination, before

15 infection, after vaccination, and/or after infection. For example, a sample can be obtained days, weeks, months and/or years after the first or second dose of the vaccine.

The invention is illustrated by the following non-limiting examples.

Examples

20 Example 1: Point-of-care rapid quantification of SARS-CoV-2 neutralizing antibody titer Summary

Neutralizing antibody (NAb) titer is a key biomarker of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but point-of-care methods for assessing NAb titer are not widely available. Here, we present a lateral flow

25 assay that captures receptor binding domain (RBD) that has been neutralized firom binding angiotensin converting enzyme 2 (ACE2). Quantification of neutralized RBD in this assay correlates with NAb titer from vaccinated and convalescent patients. This methodology demonstrated superior performance in assessing NAb titer compared to either measurement of total anti-spike immunoglobulin G titer or quantification of the absolute reduction in

30 binding between ACE2 and RBD. Our testing platform has the potential for mass deployment to aid in determining at population scale the degree of protective immunity individuals may have following SARS-CoV-2 vaccination or infection, and can enable simple at-home assessment of NAb titer.

- 19 - Background

A major aspect of global efforts to curtail the coronavirus 19 (COVD-19) pandemic involves ensuring that the majority of individuals in the population have protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Protective

5 immunity generated after either active infection or vaccination can wane over time, resulting in loss of protection against infection and disease (Collier et al., 2021; Goldberg et al., 2021). Although both humoral and cellular immunity contribute to protective immunity against SARS-CoV-2, a well-established biomarker for protective immunity predicting susceptibility to infection is neutralizing antibody (NAb) titer (Gilbert et al., 2022; Khoury et al., 2021;

10 Krammer, 2021).

Existing point-of-care assays for anti-spike and anti-RBD antibodies have insufficient sensitivity and specificity, and are likely of limited diagnostic utility in assessing true NAb status in individuals. Creation of a field-deployable test for rapid assessment of SARS-CoV-2 NAb titer has the potential to be transformative in population-scale efforts to curb the spread

15 of SARS-CoV-2. Such a test would allow individuals to know on any given day what degree of NAb protection they have circulating in their bloodstream. Furthermore, a field-deployable test would allow for large scale studies to identify the NAb titers that confer protection against SARS-CoV-2 infection. To address this unmet need, we developed a lateral flow assay (LFA) that quantifies the amount of RBD unable to bind ACE2 due to neutralization by

20 patient plasma. This LFA demonstrates robust performance in predicting NAb titer in individuals following either SARS-CoV-2 vaccination or natural infection.

Results

To develop a methodology for assessing circulating NAb titer in a rapid manner at the

25 point of care, we reasoned that neutralization of RBD:ACE2 binding (Figures 1A and IB) should serve as a core component since ELISA-based measurement of neutralization of RBD:ACE2 binding is a validated metric correlating with NAb titer. However, a weakness of relying on solely measuring a reduction in binding signal is the potential for the assay to have limited dynamic range. This is overcome in laboratory-based assays by testing serial dilutions

30 of a sample, but this approach is incompatible with point-of-care and at-home testing. To address this issue, we engineered a testing method that produces a positive signal in the presence of NAbs; this positive signal also facilitates greater ease of use and interpretation, which are paramount for successful implementation of a diagnostic test in the at-home setting

- 20 - (Murray and Mace, 2020). In this method, RBD molecules neutralized by NAbs are unable to bind ACE2 and are subsequently captured downstream in an LFA (Figure 2A).

We first generated two 293T cell lines stably secreting either recombinant RBD or a fusion protein consisting of ACE2 ECD linked with a cellulose-binding domain (hereafter

5 referred to as ACE2-CBD) for attachment to a cellulose or nitrocellulose test strip (Miller et al., 2018). Both protein constructs possessed affinity tags and could be generated with good purity (Figures 1C and 5). To validate that recombinant RBD and ACE2-CBD could bind to each other, varying amounts of ACE2-CBD were spotted onto cellulose strips, incubated with soluble RBD, and the amount of RBD bound to each ACE2-CBD spot was detected using an

10 anti-RBD monoclonal antibody (CR3022) (Huo et al., 2020). These experiments demonstrated that RBD could bind to cellulose-immobilized ACE2-CBD and that binding was blocked by a commercial recombinant monoclonal NAb isolated from a patient naturally infected with SARS-CoV-2 (Figures ID and IE).

To determine if RBD:ACE2 binding could occur in a lateral flow format, we next

15 spotted ACE2-CBD on a cellulose test strip and applied RBD diluted in pre-pandemic human plasma to one end of the test strip (Figure IF). After 15 min of lateral flow runtime, we detected RBD bound to ACE2 using the CR3022 monoclonal anti-RBD antibody. As shown in Figures 1G and 1H, RBD could bind ACE2 in a lateral flow' format, and that binding was blocked when RBD was pre-mixed with the commercial monoclonal NAb.

20 We then generated prototype LFA test cassettes comprised of a nitrocellulose strip containing a line of immobilized ACE2-CBD for capturing non-neutralized (infectious) RBD followed by a line of recombinant protein A for capturing neutralized RBD bound by NAb. In this LFA, a patient blood or plasma sample is diluted, briefly incubated with a polyester conjugate pad impregnated with colloidal gold-labeled RBD, and applied to the test strip.

25 After 20 min of lateral flow across the test strip, the ratio of gold signal at the neutralized line divided by the gold signal at the infectious line (neutralized/infectious [N/I] ratio) is measured (Figure 2A) and should correlate with NAb titer. To test this, we obtained 18 serial plasma samples from one patient before and after initial mRNA vaccination with the two- dose BNT162b2 series and 16 serial plasma samples from one patient before and after the

30 initial two-dose mRNA-1273 series. Analysis of these samples using our LFA demonstrated that the N line signal became visually apparent 2 weeks following the second dose of vaccination (Figure 2B). We quantified the signal at both the N and I lines and calculated the N/I ratio as a measure of neutralizing activity (Figures 2C and 2D). We observed a marked increase in the N/I ratio beginning 2 weeks after completing vaccination, followed by a

- 21 - decline over the course of the subsequent 4 months. We also assessed these samples with an ELISA detecting total anti-spike immunoglobulin G (IgG) antibody (Amanat et al., 2020a; Lee et al., 2020), and measured neutralization 50% inhibitory dilution (ID50) titers using pseudoviral neutralization assays. The post-vaccination kinetics of neutralization ID50 titer

5 were similar to the kinetics of the N/I ratio (Figures 2C and 2D).

To assess NAb titer following natural infection, additional plasma samples from 93 unique patients either previously infected with SARS-CoV-2 earlier than December 2020 (n = 63) or with no known exposure to SARS-CoV-2 as of December 2020 (n = 30) were obtained, analyzed with our LFA, and had anti-spike IgG and neutralization ID50 titers

10 measured. We then evaluated the ability of our LFA to predict a range of NAb titers. For N/I ratios from 0.0 to 0.7 increasing by increments of 0.001 and anti-spike IgG levels from 0 to 2,430 mg/dL increasing by increments of 5, we calculated the sensitivity and specificity for detecting neutralization ID50 titers of 1:100, 1:200, 1:300, 1:500, 1:700, and 1:1,000 in the 127 total samples assessed in our study, including samples from post vaccination and post¬

15 infection patients and patients with no known history of vaccination or infection. Receiver operating characteristic area under the curve (ROCAUC) analysis demonstrated that the diagnostic performance of our LFA achieved AUCs of greater than 0.85 for all titers assessed (Figure 3A). Compared with the anti-spike IgG ELISA, ROC-AUC analysis for all titers assessed in our study demonstrated a larger AUC for our LFA (Figure 3B). Since quantifying

20 loss of RBD:ACE2 binding signal has also been proposed as a potential surrogate measure of NAb titer (Chiu et al., 2022; Huang et al., 2022; Kongsuphol et al., 2021; Lake et al., 2021; Wang et al., 2021 ), we examined the performance of this alternative approach by quantifying only loss of signal at the I line in our LFA and compared this with measuring the N/I ratio. For all six neutralization ID50 titers assessed, use of the N/I ratio was superior to measuring

25 only loss of signal at the I line (Figures 3B).

Finally, we produced a second-generation version of our LFA with greater compatibility for use in a point-of-care or at-home setting. This version has a third line (control) consisting of immobilized poly-L-lysine located distal to the I and N lines. Poly-L- lysine will capture free colloidal gold particles flowing to the end of the strip, indicating if

30 lateral flow was successful (Figure 4A). This version also can use whole blood as the patient sample (Figure 4B). We envision that the workflow for point-of-care or at-home use would be as depicted in Figure 4C. Each test kit would contain a microcapillary tube that holds an exact volume and a finger-prick lancet (both similar to those used in certain blood glucose monitoring platforms). Each test kit would also contain a tube pre-filled with an exact volume

- 22 - of buffer, a polyester conjugate pad impregnated with RBD and colloidal gold, and a dropper cap to seal the tube. An exact volume of finger-prick blood (on the order of less than 10 mL) w'ould be collected in the microcapillary tube, which would then be placed along with the impregnated pad into the buffer-filled tube. The tube would then be sealed with the dropper

5 cap. The components would then be mixed and incubated before adding a precise number of drops onto the lateral flow strip, similar to at-home SARS-CoV-2 antigen test kits. After lateral flow for a defined time period, both qualitative and quantitative interpretation are possible. For qualitative interpretation, the cassette casing can be constructed to cover up the I line, leaving only visualization of the N and control lines, with interpretation similar to

10 SARS-CoV-2 antigen tests: appearance of two lines indicates presence of NAbs, and appearance of only the control line indicates lack of NAbs. For quantitative interpretation, the cassette casing will allow visualization of all three lines, and a smartphone application (“app’ ’) will use the phone’s existing camera to take a picture of the strip, quantify N and I tine pixel density, and report either the calculated NAb titer or an interpretation of the level of

15 NAb titer (e.g., none, low, moderate, high).

DISCUSSION

Our LFA is a tool for rapid quantification of the capability of patient plasma or blood to neutralize the RBD:ACE2 interaction. Given the diagnostic performance of our assay in

20 predicting SARS-CoV-2 NAb titer, it has the potential to make rapid assessment of NAb titer widely available without the need for centralized laboratory' processing or specialized equipment and personnel. Although our use of only the RBD portion of the SARS-CoV-2 spike protein does not assay for NAbs that neutralize spike through binding of non-RBD epitopes, we benchmarked our test against pseudoviral neutralization assays that do assess for

25 NAbs binding to non-RBD epitopes. Thus, our approach standardizing N/I signal thresholds against pseudoviral neutralization strives to take into account potential limitations from using RBD only. From a diagnostic methodology standpoint, our data demonstrate that for SARS- CoV-2 Nab titer, quantification of neutralized RBD with a positive signal increases test performance compared with assaying loss of RBD:ACE2 binding signal. This superior

30 performance may be due to our developing a design in which the N line signal is normalized to the 1 line signal, thereby helping control for run-to-run assay variability that will undoubtedly exist in point-of-care and at-home settings. This methodology' may potentially also be relevant to other infectious diseases with well-characterized epitopes for binding to surface receptors.

- 23 - As our LFA platform is compatible with existing optical capture of signal intensity using smartphone technology (Parker et al., 2020; Urusov et al., 2019; Wood et al., 2019), creation of a smartphone ‘‘app^?? specific to the final version of our assay would allow quantitative interpretation at the point of care or in the at-home setting. Additionally, because

5 this LFA platform incorporates a recombinant RBD protein that can be modularly substituted for the RBD of a SARS-CoV-2 spike protein variant of interest (VOI), our assay should be adaptable to VOIs so long as the RBD or spike protein of the variant can be quickly produced at scale in a recombinant fashion (Argentinian AntiCovid, 2020; Dalvie et al., 2021; Pino et al., 2021; Pollet et al., 2021; Zang et al., 2021). Future studies determining NAb signal in our

10 LFA following infection with SARS-CoV-2VOIs will be important, as new variants are likely to continue appearing, but orthogonal studies identifying what neutralization titers against the original SARS-CoV-2 strain are required to protect against VOIs will also be helpful in this regard (Ai et al., 2022; Evans et al., 2022a, 2022b). Finally, as this LFA reports a quantitative assessment of NAb titer, we believe it will be usefill as a cost-effective, easily

15 distributable assay in large-scale studies to define the degree of RBD:ACE2 interaction inhibition that correlates with protection from SARS-CoV-2 infection. A consensus in the field is currently lacking for this significant question regarding SARS-CoV-2 immune responses (Krammer, 2021). Once protective titers have been defined, this LFA can contribute to population-scale serial monitoring efforts to determine the time point at which

20 individuals in the general population, or high risk occupations, lose protective immunity and benefit fiom repeat vaccination.

Methods

Human sample acquisition and processing

25 Post-vaccination human blood samples w^rere collected in accordance with a protocol approved by the Boston Children’s Hospital Institutional Review Board. De-identified plasma samples from human subjects previously infected with SARS-CoV-2, or with no known previous SARS-CoV-2 exposure, were purchased fiom commercial vendors BioIVT and Ray Biotech. Human plasma samples were handled in accordance with biosafety

30 protocols in compliance with regulations of Boston Children’s Hospital. Following collection of blood samples in heparinized collection tubes fiom post-vaccination subjects, samples were centrifuged at 2,000 x g for 10 minutes at 4 degrees C, and plasma was collected, aliquoted and stored at -80 degrees C. Upon receipt of commercially purchased human

- 24 - plasma, samples were aliquoted and stored at 80 degrees C. All assays performed in this study utilized plasma thawed only once.

Generation of stable cell lines

Lentiviral constructs for creation of stable cell lines expressing the RBD and ACE2

5 ectodomain constructs were created using apHAGE2-based vector (Murphy et al., 2006) for constitutive expression of the transgene using a cytomegalovirus (CMV) promoter flanked by a mini ubiquitous chromatin opening element (miniUCOE) (Muller-Kuller et al., 2015) and a super core promoter 3 (SCP3) elements (Even et al., 2016). The vector also incorporates an IRES-blasticidin S deaminase-T2A-EGFP cassette downstream of the transgene for selection

10 using blasticidin and fluorescence activated cell sorting (FACS) based on EGFP expression level. Constructs were assembled using Gibson Assembly (Gibson et al., 2009) with the NEBuilder HiFi DNA assembly kit (New England Biolabs). A lentiviral construct for an RBD construct C-terminally tagged with His8, FLAG, StrepTag2, ALFA (Gotzke et al., 2019), and GCN4 (Tanenbaum et al., 2014) tags was assembled from tire RBD sequence PCR

15 amplified from pCAGGS-spikeRBD, while the sequences for the C-terminal tags were PCR amplified from gBlocks (Integrated DNA Technologies) codon optimized for expression in human cells. A lentiviral construct for the ACE2 ectodomain (residues 1-739) C-terminally tagged with His8, FLAG, StrepTag2 and cellulose binding domain (CBD) was assembled from fragments PCR amplified from codon-optimized gBlocks (Integrated DNA

20 technologies).

Mammalian cells lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics in a humidified incubator at 37°C with 5% CO2. Lentiviruses were produced in HEK293T cells, by calcium phosphate co¬

25 transfection (CalPhos mammalian transfection kit, Clontech Laboratories) of packaging vectors (pCMV-VSV-G envelope and pCMV-dR8.2 dvpr) together with either the RBD- or ACE2 ectodomain-expressing lentiviral construct. Virus-containing media were filtered through 0.45 pm filters and used to transduce HEK293T cells. Stably transduced cells were selected with 10 jig/mL blasticidin and sorted for high expressers by FACS using the EGFP

30 expression level as a proxy for expression of the transgene.

Purification of recombinant proteins

Media supernatant was collected from the stable cell lines every other day for two weeks. The supernatant was stored at 4 degrees Celsius until the end of the two-week period

- 25 - when it was then centrifuged at 700 x g for 5 minutes to clear any cellular debris. The spun down supernatant was then sterile filtered into a sterile bottle. Using an AKTA start system, a column filled with ImL of sedimented Strep-Tactin (2-1201-010, Neuromics Antibodies, Edina, MN) resin was attached and washed with 15 mL of 50 mM 4-(2-hydroxyethyl)-l-

5 piperazineethanesulfonic acid (HEPES) buffer. The media supernatant was run through the column at a speed of ImL/min overnight at 4 degrees Celsius. The column was washed for 15 minutes with 15 mL of wash buffer (50 mM HEPES) and was then switched out for elution buffer (50mM biotin HEPES). Elution buffer was run through the column until the UV absorbance returned to the level it was prior to elution. The elution product was then

10 concentrated via 100,000 kDa molecular weight cut-off (MWCO) centrifuge tubes. Concentration was then measured by bicinchoninic acid (BCA) assay analysis. Purity was analyzed by running 2 pL & 5 pL of elute product on an sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) gel then stained with Coomassie blue.

15 Dot blot assessment of RBD-ACE2 interaction

Serial dilutions of the recombinant ACE2-CBD were made in the same buffer it was eluted in to yield 250 ng, 500 ng, and 1000 ng of ACE2-CBD per 3 pL of solution. The serial dilutions were applied to cellulose membrane (Whatman #2) cut to size and incubated at 37 degrees Celsius for 2 hours. The membranes were then blocked in 3% bovine serum albmin

20 (BSA) for 1 hour at 25 degrees Celsius. Primary incubation solutions were made in 3% BSA buffer with RBD at a concentration of 1 pg/mL (except for the no RBD condition) and increasing NAb (0.05 pg, 0.5 pg, 5 pg NAb) all solutions were 1 mL final volume. The NAb used was a SA RS - Co V -2 Neutralizing Antibody (A19215, ABclonal, Woburn, MA). The primary incubation solutions were all incubated on ice for 15 minutes. Blocking buffer was

25 discarded, primary solution was added and set to incubate at 4 degrees Celsius for 1 hour. Blots were then washed 3 times for 5 minutes each with Tris-buffered saline with 0.1% Tween-20 (TBST) buffer. Primary antibody (Mouse lgG2A-modified Anti-COV1D-19 & SARS-CoV S glycoprotein [CR3022 (ter Meulen et al., 2006)], Absolute Antibody) solution was applied at 1 : 1000 dilution in TBST buffer and incubated at 4 degrees Celsius overnight.

30 Blots were washed again with TBST, three times, 5 min each. Next, anti-mouse IgG- horseradish peroxidase conjugated secondary antibody (#7076, Cell Signaling Technologies) was diluted into 3% BSA TBST buffer at 1:5000, applied to blots and incubated at 25 degrees Celsius for 1 hour. Membranes were then washed again with TBST, 3 times, 5 min each. ECL reagent (ThermoFisher) was mixed and applied to each blot for 5 minutes, then blotted

- 26 - off. Blots were then all exposed and imaged together via Bio Rad ChemiDoc XRS+ system on auto-exposure. Integrated density analysis of dot blots was performed on Image! according to the Image! NIH Dot Blot Analysis tutorial.

5 Pilot lateral flow assessment of RBD-ACE2 interaction

Whatman #2 cellulose membrane was cut it into thin strips of 0.5 cm by 8.5 cm. At 2 cm up from the bottom of the strip 1 pg of ACE2-CBD was applied and the strips were then dried at 37 degrees Celsius for 2 hours. The strips were then blocked with 3% BSA TEST solution for 1 hour at 25 degrees Celsius. Afterwards, the strips were dried at 37 degrees

10 Celsius for 1 hour once more. During this time, the samples were prepared in saline with fleshly thawed pre-pandemic collected plasma (70039.6, Stem Cell Technologies) at a 1:50 ratio. All samples excluding the no RBD control had 1 pg RBD present in solution. SARS- CoV-2 NAb was added to three samples in increasing concentration: 0.05 pg, 0.5 pg, 5 pg as well as 5 pg added to the no RBD control. Samples were all 70 pL in volume and incubated

15 on ice for 1 hour. The dried strips were laid on a 10 degree downward slope with the ACE2 end being the highest in elevation. Samples were added dropwise to the bottom of the strips and left to run for 15 minutes. The strips were then washed three times with TEST for 5 minutes each. Primary' antibody (Mouse IgG2A-modified Anti-COVID-19 & SARS-CoV S glycoprotein [CR3022], Absolute Antibody) solution was made at a 1: 1000 ratio in 3%

20 bovine serum albumin TBST buffer and added to the strips to incubate at 4 degrees Celsius overnight. The strips were washed three times again with TBST 5 minutes each wash. Antimouse IgG- horseradish peroxidase conjugated secondary antibody (#7076, Cell Signaling Technologies) was diluted in 3% BSA TBST buffer at a 1 :5000 dilution and applied to the strips to incubate for 1 hour at 25 degrees Celsius. After, the strips were washed three times

25 again for 5 minutes each with TBST. Enhanced chemiluminescence (ECL) reagent was then mixed and applied to strips for 5 minutes before blotting off solution and imaging the strips on a Bio-Rad ChemiDoc set to auto-exposure. Integrated density analysis of lateral flow strips was performed on Image! according to the Image! NIH Dot Blot Analysis tutorial.

30 Lateral flow assay cassette construction

Each lateral flow assay cassette is composed of an Ahlstrom 8964 fiber glass sample pad (22 x 3.9 mm; Ahlstrom-Munksjo, Finland), a Sartorius CN95 nitrocellulose membrane (25 x 3.9 mm; Sartorius, Germany) and an Ahlstrom 222 absorbent “sink” pad (20 x 3.9 mm; Ahlstrom-Munksjo, Finland), which were assembled sequentially onto a plastic-backed

- 27 - support card, such that there was an overlap of 2 5 mm between each pair of pads. Briefly, the nitrocellulose membrane and the sink pad were assembled first, after which the nitrocellulose membrane was striped twice, first with 0.47 mg of ACE-CBD, which served as the Infectious line, and then with 1.0 mg of recombinant protein A, which served as the

5 Neutralized line (Figure 3A). The two proteins were dispensed 9 mm apart, such that the ACE2-CBD and the recombinant protein A were striped 34 mm and 25 mm, respectively, from the outer edge of the sink pad. The striped nitrocellulose membrane was then dried at 50°C for 2 days. Finally, the sample pad was added, and the assembled strip was equipped with a Lohmann GL-422 clear cover-stock (45 x 3.9 mm; Lohmann Technologies,

10 Kentucky'), loaded into a plastic shell, and stored in a vacuum-sealed foil pouch under anhydrous conditions for future use.

Lateral jlow assay protocol

Frozen aliquots of the commercial and post-vaccination plasma samples were first

15 thawed at room temperature and then placed on ice. Each plasma aliquot was then vortexed briefly for approximately 10 seconds, after which 1 pL of each sample was diluted 1:800 into room temperature running buffer (10 mM Tris-HCl Buffer with 1 mM ethylenediaminetetraacetic acid (EDTA), 5% Sea-Block, 3% Triton X305, 1% spike/v sodium chloride and 0.1% Bronidox; Catalloid Products, Inc.) in a 1.5 mL Eppendorf tube.

20 The diluted plasma samples were then mixed thoroughly by vortexing and incubated at room temperature. Following a 10 -minute incubation period, 150 pL of each diluted plasma sample were then transferred to a separate, clean 1.5 mL Eppendorf tube along with a single 5 mm circular-punched Ahlstrom 6615 colloidal gold pad to which recombinant RBD protein was conjugated (Catalloid Products, Inc). The tubes were subsequently placed in a

25 microcentrifuge rack and left to incubate for 10 minutes at 25 degrees Celsius with continuous shaking at 200 rpm on a Model G2 Gyratory Shaker (New Brunswick Scientific Co., New Jersey). 75 pL of each RBD/colloidal gold-incubated plasma sample were then added to tire sample well of a lateral flow' assay test cassette and allowed to travel up the nitrocellulose membrane test strip by hydrodynamic force and capillary action. After 10

30 minutes, each sample pad was washed by adding 20 pL of running buffer (Catalloid Products, Inc.). Assay signals were allowed to develop for another 10 minutes, after which the test cassettes were placed in a shallow, ceiling-less Styrofoam box with constant, uniform overhead lighting and images immediately captured using a Fujifilm X-E2S digital camera (Fujifilm, Japan) at constant focus, aperture, and shutter speed settings. The intensities of the

- 28 - signals at the infectious (control) and the neutralized (test) lines in the image taken from each test cassette were then quantified in ImageJ according to the Image! NIH Dot Blot Analysis tutorial, without any background correction. The ratio of the intensities of the neutralized line signal and the infectious line signal (N/I ratio) was calculated for each sample to determine

5 the neutralizing antibody titer.

Pseudoviral neutralization assay

ACE2 expressing HEK293 cells (#79951, BPS Bioscience, San Diego CA) were cultured in growth medium IN culture media (#79801, BPS Bioscience, San Diego CA) in

10 96-well white with transparent bottom plates. The day before the assay cells were seeded at a density of 10,000/well in 100 pL of media and left to incubate at 37 degrees Celsius overnight. The following day assay samples were prepped by first thawing plasma samples on ice then diluting them at either 1 : 10, 1 : 100 or 1 : 10000 in media with 1 pL of spike pseudotyped lentivirus with a luciferase reporter (#79942, BPS Bioscience, San Diego CA)

15 up to 10 pL. After a 30-minute incubation period the plasma-pseudotype samples were added to the cells. The cells were then left to incubate for 48 hours. After 48 hours, the ONE-Step luciferase reagent (#60690, BPS Bioscience, San Diego CA) was prepared per protocol and luminescence was measured on a Tecan Infinite m200 pro plate reader. Neutralization IDso titers were calculated using the “One-Site Fit LogICSO” regression function in GraphPad

20 Prism 8.0

Anti-spike IgG ELISA

Plates (Nunc MaxiSorp) were coated overnight at 4°C with recombinant spike protein SI subunit (Biolegend) at a concentration of 1 pg/mL. Wells were subsequently blocked

25 with phosphate buffered saline containing 5% bovine serum albumin. After washing, human plasma samples (1 :200 dilution) were added and the plates were incubated fbr 2 hours at 25 degrees Celsius. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Southern Biotech; 1 : 1000 dilution) was added after washing and the plates were developed using TMB substrates (Biolegend). Absorbance at OD600 was measured using a Synergy HTX Reader

30 (BioTek). Antibody units were determined based on a standard curve constructed using commercially available monoclonal human anti-spike protein antibodies (Biolegend, clone AM001414).

Sensitivity and Specificity calculations

- 29 - To assess and compare the performances of the lateral flow assay and the ELISA based anti-spike IgG test at identifying plasma samples with specific neutralization titers, the sensitivity and specificity rates for a range of signal threshold values were determined for both tests in R (Version 3.5.2). This was done for neutralization IDso titer cutoffs of 1: 100,

5 1:200, 1:300 1:500 and 1:1000, with specificity and sensitivity values being calculated for N/I ratios from 0 to 0.5 increasing in increments of 0.001 for the lateral flow assay, and for anti-spike IgG levels from 0 to 2400 mg/dL in increments of 5 mg/dL. The optimal signal threshold that would maximize both sensitivity and specificity for each test for a given neutralization titer cutoff was then determined using Excel.

10 To calculate the sensitivity and specificity rates of either test for a given signal threshold, the following formulae were employed, using the specific pseudoviral neutralization assay IDso titer cutoff:

Sensitivity Rate

_ Number of Samples with Test Signal > Threshold & Neutralization IC₅₀ Titer > Cutoff

^— Total Number of Samples with Neutralization IC_5O Titer > Cutoff

15

Specificity Rate

_ Number of Samples with Test Signal < Threshold & Neutralization IC₅₀ Titer < Cutoff

- Total Number of Samples with Neutralization IC_S0 Titer < Cutoff

To assess the diagnostic performance of the lateral flow assay test for measuring the loss of binding signal at the infectious line, the sensitivity and specificity values were

20 calculated using just the integrated densities from the infectious line from the lateral flow assay test cassettes for the five different neutralization IDso titers. These calculations were made for integrated density values between 15000 and 21000 in increments of 20 using the following formula:

Sensitivity Rate

Number of Samples with Infectious Signal < Threshold & Neutral. IC_S0 Titer > Cutoff

25 ~ Total Number of Samples with Neutralization IC_5O Titer > Cutoff

Specificity Rate

_ Number of Samples with Infectious Signal > Threshold & Neutral. IC_S0 Titer < Cutoff

” Total Number of Samples with Neutralization IC_S0 Titer < Cutoff

The optimal integrated density threshold that would maximize sensitivity and

30 specificity of the test was determined in Excel.

Receiver Operating Curve Generation

- 30 - The calculated sensitivity and specificity values were used to generate receiver operating characteristic (ROC) curves fbr both tests for IDso titer cutoffs of 1 : 100, 1 :200, 1 :300 1 :500 and 1 : 1000. This was achieved by plotting the sensitivity' rates as a function of the false positive (1 -specificity) rates. The area under each ROC curve was determined using

5 the receiver operating curve functionality in GraphPad Prism 8.

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- 34 - All references, articles, patent applications, patent publications and patents are incorporated herein by reference in their entirety. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein

5 without departing from the scope of the invention encompassed by the appended claims.

- 35 -

Claims

CLAIMS What is claimed is:

1. A lateral flow test strip for detecting the presence or titer of neutralizing antibodies

5 (nAbs) to SARS-CoV-2 in a biological sample from a patient, wherein the test strip comprises: a sample application region; a control line comprising an immobilized extracellular domain of the human angiotensin II converting enzyme type 2 (ACE2) receptor; and a test line comprising an immobilized human immunoglobulin G (IgG) binding agent;

10 wherein the control line is located downstream from the sample application region and the test line is located downstream from the control line, or wherein the test line is located downstream from the sample application region and the control line is located downstream from the test line; and wherein the strip is configured such that the presence of nAbs is detected by

15 detecting the first detectable label captured at the test line, and/or wherein the strip is configured such that the titer of nAbs is determined by calculating the ratio of the amount of first detectable label captured at the test line to the amount of first detectable label captured at the control fine.

20 2. The lateral flow test strip of claim 1, wherein the strip further comprises a conjugation region, wherein the conjugation region comprises a SARS-CoV-2 antigen conjugated to a first detectable label, and wherein the conjugation region is downstream of the sample application region and upstream of the control fine and the test line.

25

3. The lateral flow test strip of claim 1 or 2 wherein the control line is located downstream from the sample application region and the test line is located downstream from the control line.

30 4. The lateral flow test strip of any one of claims 1-3 wherein the IgG binding agent comprises an IgG binding fragment selected from the group consisting of IgG binding fragment of protein A, an IgG binding fragment of protein G, an IgG binding fragment of protein A/G, an IgG binding fragment of protein L, an IgG binding fragment of protein M, an IgG binding fragment of protein Z, an IgG

- 36 - binding fragment of protein L/A, and IgG binding fragment of protein L/G, or wherein the IgG binding agent is a protein selected from the group consisting of protein A, protein G, protein A/G, protein L, protein M, protein Z., protein L/A, and protein L/G.

5

5. The lateral flow test strip of claim 4, wherein the protein is recombinant.

6. The lateral flow test strip of claim 5, wherein the protein is recombinant protein A.

10 7. The lateral flow test strip of any one of claims 1-6, wherein the IgG binding agent comprises an IgG binding fragment of protein A.

8. The lateral flow test strip of any one of claims 1-7, wherein the IgG binding agent is poly-l-lysine.

15

9. The lateral flow test strip of any one of claims 1-8, wherein the IgG binding agent is an anti-lgG antibody.

10. The lateral flow test strip of any one of claims 2-9, wherein the first detectable label

20 is visible to the naked eye.

11. The lateral flow test strip of claim 10, wherein the first detectable label is a noble metal nanoparticle.

12. The lateral flow test strip of claim 11, wherein the first detectable label is a gold nanoparticle.

25 13. The lateral flow test strip of any one of claims 1-12, wherein the strip comprises treated biological sample in the sample application region or downstream therefrom.

14. The lateral flow test strip of any one of claims 2-13, wherein the SARS-CoV-2 antigen comprises the membrane protein or fragment thereof, the spike protein or fragment thereof, the envelope protein or fragment thereof or the nucleoprotein or

30 fragment thereof.

15. The lateral flow test strip of claim 14, wherein the SARS-CoV-2 antigen comprises all or a portion of the spike protein.

- 37 -

16. The lateral flow test strip of claim 14, wherein the SARS CoV 2 antigen comprises all or a portion of the receptor binding domain (RBD) of the spike protein.

17. The lateral flow test strip of claim 16, wherein the SARS-CoV-2 antigen comprises all or a portion of the receptor binding domain (RBD) of the spike protein and does

5 not comprise the entire spike protein.

18. The lateral flow test strip of claim 16, wherein SARS-CoV-2 antigen is the RBD of the spike protein.

19. The lateral flow test strip of any one of claims 14-18, wherein the SARS-CoV-2 antigen is recombinant.

10 20. The lateral flow test strip of any one of claims 14-18, wherein the SARS-CoV-2 antigen is conjugated to the first detectable label comprises a gold nanoparticle.

21. The lateral flow test strip of any previous claim, wherein the lateral flow test strip comprises nitrocellulose or cellulose.

22. The lateral flow test strip of claim 21, wherein the ACE2 receptor extracellular

15 domain is a fusion protein comprising the ACE2 receptor extracellular domain fused to a cellulose binding domain and wherein the fusion protein is immobilized to the test strip by the binding of the cellulose binding domain to the test strip.

23. The lateral flow test strip of any preceding claim, wherein the test strip is a point-of- care lateral flow test strip.

20 24. The lateral flow test strip of any preceding claim, comprising an one or more additional control lines.

25. The lateral flow test strip of claim 24, wherein the one or more additional control lines comprises a second control line, wherein the second control line comprises immobilized poly-L-lysine,.

25 26. The lateral flow test strip of any preceding claim, wherein the control line is located downstream from the sample application region, the test line is located downstream from the control line, and a second control line comprising immobilized poly-L- lysine downstream of the test line e.

- 38 -

27. A diagnostic kit comprising (a) the lateral flow test strip of any one of claims 1 24; and (b) a SARS-CoV-2 antigen labeled with a first detectable label.

28. The diagnostic kit of claim 27, further comprising a sample collection device for a biological sample.

5 29. The diagnostic kit of claim 28, wherein the sample collection device is a microcapillary tube.

30. The diagnostic kit of any one of claims 27-29, wherein the kit comprises an absorbent pad impregnated with the SARS-CoV-2 antigen.

31. The diagnostic kit of claim 30, further comprising a running buffer and optionally

10 comprising a chase buffer.

32. The diagnostic kit of any preceding claim, wherein the first detectable label is a gold nanoparticle.

33. The diagnostic kit of any preceding claim, wherein the SARS-CoV-2 antigen comprises the membrane protein or fragment thereof, the spike protein or fragment

15 thereof, the envelope protein or fragment thereof or the nucleoprotein or fragment thereof.

34. The diagnostic kit of any preceding claim, wherein the SARS-CoV-2 antigen comprises all or a portion of the spike protein.

35. The diagnostic kit of any preceding claim, wherein the SARS-CoV-2 antigen

20 comprises all or a portion of the receptor binding domain (RBD) of the spike protein.

36. The diagnostic kit of any preceding claim, wherein the SARS-CoV-2 antigen comprises all or a portion of the receptor binding domain (RBD) of the spike protein and does not comprise the entire spike protein.

25 37. The diagnostic kit of any preceding claim, wherein SARS-CoV-2 antigen is the RBD of the spike protein.

38. The diagnostic kit of any preceding claim, wherein the SARS-CoV-2 antigen is recombinant.

- 39 -

39. The diagnostic kit of any preceding claim, wherein the first detectable label comprises a gold nanoparticle

40. A method of detecting the presence or titer of neutralizing antibodies (nAbs) to SARS-CoV-2 in a biological sample of a patient, comprising the steps of:

5 (a) contacting the biological sample with a SARS-CoV-2 antigen for a time sufficient for binding of nAbs present in the sample to bind the SARS-CoV-2 antigen thus forming a treated sample, wherein the SARS-CoV-2 antigen is conjugated to a first detectable label; wherein the contacting step occurs before or after application to a sample application region;

10 (b) applying the sample or the treated sample to the sample application region of the lateral flow test strip of any preceding claim so as to permit flow of the test sample from tire sample application region to the control line and to the test line; and

(c) detecting the first detectable label at the test line and optionally detecting the first detectable label at the control line;

15 wherein the presence of the first detectable label at the test line indicates the presence of nAbs and/or wherein the titer of nAbs is determined by calculating the ratio of the amount first detectable label captured at the test line to the amount of first detectable label captured at the control line.

20 41. The method of claim 38, wherein the first detectable label is a gold nanoparticle.

42. The method of any preceding claim, comprising measuring the amount of label at the test line and optionally the control line and wherein the presence of the first detectable label at the test line indicates the presence of nAbs and/or wherein the titer of nAbs is determined by calculating the ratio of the amount of first detectable

25 label captured at the test line to the amount of first detectable label captured at the control line.

43. The method of any preceding claim, wherein the amount of first detectable label at the test line and optionally the control line is measured using a handheld computing device, optionally, a smartphone camera.

30 44. The method of any one claims 38-41, wherein the sample is contacted with the SARS-CoV-2 antigen after application to the sample application region.

- 40 -

45. The method of any one claims 38 41, wherein the sample is contacted with the SARS-CoV-2 antigen before application to the sample application region.

46. The method any one claims 38-41, wherein the sample is contacted with an absorbent pad impregnated with the SARS-CoV-2 antigen before application to the

5 sample control region.

47. The method of claim 44, wherein the absorbent pad is incubated with running buffer to form the treated sample.

48. The method of any preceding claim, wherein the SARS-CoV-2 antigen comprises the membrane protein or fragment thereof, the spike protein or fragment thereof, the

10 envelope protein or fragment thereof or the nucleoprotein or fragment thereof.

49. The method of any preceding claim, wherein the SARS-CoV-2 antigen comprises all or a portion of the spike protein.

50. The method of any preceding claim, wherein the SARS-CoV-2 antigen comprises all or a portion of the receptor binding domain (RBD) of the spike protein.

15 51. The method of any preceding claim, wherein the SARS-CoV-2 antigen comprises all or a portion of the receptor binding domain (RBD) of the spike protein and does not comprise the entire spike protein.

52. The method of any preceding claim, wherein SARS-CoV-2 antigen is the RBD of the spike protein.

20 53. The method of any preceding claim, wherein the SARS-CoV-2 antigen is recombinant.

54. The method of any preceding claim, wherein the first detectable label comprises a gold nanoparticle.

55. The method of any preceding claim, wherein the biological sample is a blood

25 sample, a serum sample, a saliva sample, or an abrasive gum swab.

56. The method any preceding claim, wherein the sample is collected with a microcapillary tube.

57. The method of any preceding claim, wherein the method is conducted at the point of care.

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58. The method of any preceding claim, wherein the accuracy of the results is about 90% or greater.

59. The method of any preceding claim, wherein the test strip provides results in less than about 30 minutes.

5 60. The method of any preceding claim, wherein the method is used to monitor antibody titer after vaccination or after SARS-CoV-2 infection.

61. The method of claim 58, comprising identifying said patient as being in need of a vaccine booster shot if the level of nAbs in the sample is below a predetermined threshold level.

10 62. The method of claim, 59, further comprises administering a vaccine booster to said patient identified as being in need of a vaccine booster shot.

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