Neutralizing antibody testing and treatment

BACKGROUND

SARS-CoV-2 is a β coronavirus and causes COVID-19, an acute respiratory infectious disease. Humans are generally susceptible. Individuals infected with SARS-CoV-2 are the main source of infection, but infected people who are asymptomatically infected are also a source of infection. Based on the current epidemiological investigation, the incubation period is 2 to 14 days, with a median of 5 days. The main manifestations of COVID19 include fever, fatigue and dry cough. Nasal congestion, runny nose, sore throat, myalgia and diarrhea may also be present.

People who've recovered from COVID-19 have antibodies to the virus in their blood. Plasma prepared from these individuals is referred to as COVID19 convalescent plasma (CCP). CCP can be given to people with severe COVID-19 with the intention of boosting their ability to fight the virus.

Once someone recovers clinically and tests: (A) negative by PCR (no live virus present) and (B) positive by serology test (antibodies to SARS-Cov2 present), they may be asked if they would like to donate CCP. If they agree, they undergo plasmapheresis after which their plasma is then frozen, usually in 200 cc units.

When someone fighting COVID19 needs CCP, a unit of frozen plasma is available. No tests for antibody abundance or their ability to neutralize the virus are performed. It is assumed that CCP contains neutralizing antibodies.

However, it has been shown that some patients make high titers of neutralizing Ab, but others don't at all—even though they both recover. This means some patients get much more neutralizing Ab than they need while others don't get enough.

Because it is of high clinical interest to correlate neutralizing Ab titers to clinical outcome, there is a need for new to diagnostic methods, devices and kits for detecting neutralizing antibodies to SARS-CoV-2.

SUMMARY

Provided herein are methods for detection and measurement of neutralizing antibody levels to a coronavirus (e.g., SARS-CoV-2, and the like) in a test-specimen, said method comprising:

obtaining a test-specimen from a subject;

transferring the test-specimen to a sample well of a test-cassette, wherein the cassette further comprises a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad, wherein the sample pad comprises ACE2 or a functional fragment thereof, wherein the conjugate pad comprises a viral-ACE2-binding protein coupled to a label;

adding a buffer; and

reading the results from the test-cassette.

The invention methods are useful herein: to test pre-collected convalescent plasma form patients known to have had COVID19; to test a pre-donated sample using a drop of blood (e.g., 10 microliter drop) from a lancet finger-stick from a patient known or suspected of having been infected with COVID19; and/or as a post-vaccine companion diagnostic to determine whether and how much vaccine administration has produced neutralizing antibodies to SARS-CoV-2.

In one embodiment, the invention diagnostic method is referred to herein as the IMMUNOPASS diagnostic method. The IMMUNOPASS SARS-Cov-2 Neutralizing Antibody Rapid Test is a rapid test that utilizes a combination of SARS-COV-2 antigen coated colored particles and a modified human ACE2 protein receptor for the detection of antibodies to SARS-COV-2 in serum or plasma that block interaction of the virus with human cells expressing ACE2. IMMUNOPASS is a rapid point of care test that measures relative levels of antibodies (e.g., neutralizing antibodies referred to herein as NAbs) against Spike protein receptor binding domain (RBD) that block it from binding to ACE2 cellular receptor. Such antibodies have been shown in peer-reviewed publications to neutralize virus and will be referred to as “neutralizing antibodies”. Neutralizing antibodies may be any isotype. In certain embodiments, the invention IMMUNOPASS lateral flow test can be used for rapid detection of neutralizing antibodies to SARS-CoV-2 in plasma, serum or whole blood. “Recovered” indicates individuals have become PCR negative and may have tested positive in a COVID19 serology test for total Ig or IgG.

The invention IMMUNOPASS diagnostic test is intended for semi-quantitative measurement of neutralizing antibody levels in plasma or serum from individuals who have had recent or prior infection with SARS-CoV-2 and who have recovered from COVID19 and individuals who have received a COVID19 vaccine. The invention methods and products are useful as clinical decision-making tools for therapeutic administration of convalescent plasma for treatment of patients fighting COVID19.

Because several publications have shown that >30% of COVID19 convalescent plasma does not neutralize SARS-CoV-2 in either spike protein pseudotype or authentic SARS-CoV-2 plaque reduction neutralization assays, the IMMUNOPASS test advantageously addresses the question of whether convalescent plasma from recovered COVID19 patients contains neutralizing antibodies suitable for administration to patients actively fighting COVID19. In typical embodiments, the test should be performed with positive and negative controls. Currently, it is unknown for how long antibodies persist following infection, but the invention IMMUNOPASS methods, devices and kits provide the ability to accurately measure levels of neutralizing antibodies in convalescent plasma.

The results described herein are for the semi-quantitative measurement of antibodies which neutralize SARS-CoV-2. Antibodies to SARS-CoV-2 are generally detectable in blood several days after initial infection, although the duration of time antibodies are present post-infection is not well characterized. Individuals may have detectable virus present for several weeks following seroconversion. Detection and measurement of high levels of neutralizing antibodies may limit virus transmission and protect individuals from re-infection.

In particular embodiments, the test-specimen is whole blood, plasma or serum. In certain embodiments, the whole blood, plasma or serum is obtained from a patient either known or suspected of recovering from COVID19 disease; or known to have been vaccinated for SARS-CoV-2. In particular embodiments, the plasma is obtained using anti-coagulants such as heparin, dipotassium EDTA or sodium citrate, and the like.

In certain embodiments, wherein the test-specimen is whole blood, plasma, serum and/or saliva. In particular embodiments, the whole blood, plasma, serum or saliva is obtained from a patient either known or suspected of recovering from COVID19 disease; or known to have been vaccinated for SARS-CoV-2. In certain embodiments, ACE2 is bound directly on the sample pad, or in other embodiments, ACE2 is bound to the sample pad via a tag/anti-tag pair. In a particular embodiment, ACE2 is bound to biotin; and the sample pad is bound to streptavidin. In typical embodiments, the viral-ACE2-binding protein is an RBD.

In certain embodiments, the plasma is obtained using an anticoagulant. In yet further embodiments, the anticoagulant is selected from the group consisting of: heparin, dipotassium EDTA or sodium citrate. In particular embodiments, the label is selected from a nanoparticle, bead, latex bead, aptamer, and/or a quantum dot. In another embodiment, the conjugate pad further comprises a mixture of RBD coupled to a nanoparticle and control-antibody coupled to a nanoparticle. In one embodiment, the RBD is coupled to a gold nanoshell (GNS) and the control-antibody is a monoclonal antibody (e.g., a mouse Mab, or the like) coupled to a gold nanosphere (GNP). In particular embodiments, reading the results from the test-cassette further comprises determining the intensity of a test-line in the test-cassette compared with a reference standard. In a particular embodiment, the reference standard is a scorecard.

In certain embodiments, the level of anti-SARS-CoV-2 NAbs in the test-specimen is reported as falling within a range of pre-determined values. In a particular embodiment, the range of pre-determined values corresponds to high, moderate or low/non-neutralizing relative to three respective controls. In another embodiment, the range of pre-determined values corresponds to High (H), Moderate-High (MH), Moderate to Moderate-High (M-MH), Moderate (M), Moderate to Not Detectable (M-ND) and Not Detectable (ND).

Also provided herein are methods of determining the levels of protective neutralizing antibodies induced by a SARS-CoV-2 vaccination or infection of a particular subject, comprising:

obtaining a test-specimen from a subject, wherein the subject was previously vaccinated; or known or suspected to have been previously infected with SARS-CoV-2; and

detecting the presence and/or quantity of NAb according to methods provided herein for detection of neutralizing antibodies to SARS-CoV-2 in a test-specimen.

In certain embodiments, the subject was vaccinated or infected prior to obtaining the test-specimen in the range of: 1-365 days, 2-300 days, 3-275 days, 4-250 days, 5-225 days, 6-200 days, 7-180 days, 8-180 days, 9-180 days, 10-180 days, 11-180 days, 12-180 days, 13-180 days, and/or 14-180 days. In typical embodiments, detecting the presence of NAbs above a threshold value indicates protective antibody-based vaccination or infection.

Also provided herein are methods of identifying high-titer anti-SARS-CoV-2 NAbs samples induced by SARS-CoV-2 vaccination or infection of a particular subject, comprising:

obtaining a test-specimen from a subject, wherein the subject was previously vaccinated; or known or suspected to have been previously infected with SARS-CoV-2; and

detecting the presence and/or quantity of NAb according to methods provided herein for detection of neutralizing antibodies to SARS-CoV-2 in a test-specimen.

Also provided herein are methods of measuring neutralizing antibody levels to SARS-CoV-2 in a specimen using an electronic device, said method comprising:

scanning a code into the electronic device that identifies a test to be performed and a particular specimen to be tested;

conduct the method of detecting the presence and/or quantity of NAb according to methods provided herein for detection of neutralizing antibodies to SARS-CoV-2 in a test-specimen; and

scanning the results obtained from the test-cassette into the electronic device.

In typical embodiments, the results are processed directly on the electronic device. In particular embodiments the electronic device is a smartphone, tablet or personal computer. In other embodiments, the electronic device further connects to a database, thereby transferring the results to said database. In certain embodiments, the device connects to the database via email, WiFi, SMS, worldwide web, 4G, 5G, Bluetooth and/or USB.

Also provided herein are SARS-CoV-2 test-cassette devices, comprising a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad, wherein the sample pad and/or conjugate pad comprises ACE2 or a functional fragment thereof, and wherein the conjugate pad comprises a viral-ACE2-binding protein coupled to a label. In certain embodiments, the ACE2 is bound directly on the sample pad and/or conjugate pad; or ACE2 is bound to the sample pad and/or conjugate pad via a tag/anti-tag pair. In particular embodiments, ACE2 is bound to biotin; and the nitrocellulose membrane is bound to streptavidin. In particular embodiments, the viral-ACE2-binding protein is an RBD. In yet other embodiments, the conjugate pad further comprises a mixture of RBD coupled to a nanoparticle and control-antibody coupled to a nanoparticle. In other embodiments, the RBD is coupled to a gold nanoshell (GNS) and the control-antibody is a monoclonal antibody coupled to a gold nanosphere (GNP).

In particular embodiments, a whole-blood filter is present in lieu of the sample pad. In certain embodiments, the conjugate pad comprises a viral-ACE2-binding protein coupled to a label; and further comprises ACE2 or a functional fragment thereof. In particular embodiments, the ACE2 or functional fragment thereof is spatially separated from the viral-ACE2-binding protein. In one embodiment, the viral-ACE2-binding protein is an RBD region of a SARS-CoV-2 spike protein.

Also provided herein are SARS-CoV-2 test-cassette devices, comprising a whole blood filter, a conjugate pad, a nitrocellulose membrane and an absorbent pad, wherein the conjugate pad comprises ACE2 or a functional fragment thereof, and a viral-ACE2-binding protein coupled to a label. In certain embodiments, ACE2 is bound directly on the conjugate pad; or ACE2 is bound to the conjugate pad via a tag/anti-tag pair. In other embodiments, ACE2 is bound to biotin; and the nitrocellulose membrane is bound to streptavidin. In a particular embodiment, the viral-ACE2-binding protein is an RBD. In certain embodiments, the conjugate pad further comprises a mixture of RBD coupled to a nanoparticle and control-antibody coupled to a nanoparticle. In yet further embodiments, the RBD is coupled to a gold nanoshell (GNS) and the control-antibody is a monoclonal antibody coupled to a gold nanosphere (GNP). In yet other embodiments, the ACE2 or functional fragment thereof is spatially separated from the viral-ACE2-binding protein. In one embodiment, the viral-ACE2-binding protein is an RBD region of a SARS-CoV-2 spike protein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 shows a schematic of one embodiment of the invention IMMUNOPASS Neutralization LFA. Below each graphic is a representative image of a lateral flow strip demonstrating actual line density. Addition of non-COVID19-immune serum or plasma (top) does not block binding of RBD-beads to ACE2 resulting in the RBD-bead-ACE2 complex creating a visible line. Addition of moderate titer NAbs to the sample pad creates a weak line (middle). Addition of high titer NAbs (>1:640) blocks binding of RBD-beads to ACE2 such that no line is observed at the test location on the strip (bottom). Red control line represents capture of gold nanospheres coupled to a monoclonal antibody (e.g., a mouse Mab, or the like).

FIG. 2A shows one embodiment of an IMMUNOPASS Scorecard for measuring 3 relative levels of neutralizing antibodies in plasma or serum.

FIG. 2B shows one embodiment of an IMMUNOPASS Scorecard for measuring 4 relative levels of neutralizing antibodies in plasma or serum

FIG. 3A corresponds to the internal placement of general exemplary components of a particular embodiment of an IMMUNOPASS lateral flow strip cassette.

FIG. 3B corresponds to the internal placement of particular components of a particular embodiment of an IMMUNOPASS lateral flow strip cassette.

FIG. 4A shows a graphical representation of Applicant Table 1.

FIG. 4B also shows a graphical representation of Applicant Table 1.

FIG. 5 shows a bar graph with individual points from Table 3 depicted therein.

FIG. 6 shows box plots of LFA values by titer.

FIG. 7 shows a depiction of pipetting plasma to the sample along with buffer.

FIG. 8A shows an interpretation of results of a test strip after undergoing an invention diagnostic assay.

FIG. 8B also shows an interpretation of results of a test strip after undergoing an invention diagnostic assay.

FIG. 9A shows a printed score card next to the observation window of an invention diagnostic cartridge.

FIG. 9B shows a printed test-results score card for assessing 4 relative levels of NAbs from an invention diagnostic cartridge.

FIG. 10 shows the results of the clinical performance of the IMMUNOPASS SARS-Cov-2 Neutralizing Antibody Rapid Test (Serum/Plasma) evaluated by testing a total of 180 plasma (EDTA, ACD, heparin) clinical samples.

FIG. 11 shows the results from whole blood of evaluating vaccine-induced Nab levels.

FIG. 12 shows the result from whole blood that previous natural infection with SARS-CoV-2 does not produce high levels of NAbs, whereas a single dose of vaccine in these previously SARS-CoV-2 infected subjects produces high levels of NAbs.

FIG. 13A shows a plasma panel regarding the differences between Whole Blood vs Plasma Assay Embodiments of the invention test-cassette devices.

FIG. 13B. shows a whole blood panel regarding the differences between Whole Blood vs Plasma Assay Embodiments of the invention test-cassette devices.

DETAILED DESCRIPTION

Provided herein are methods for detection and measurement of neutralizing antibody levels to a coronavirus (e.g., SARS-CoV-2, and the like) in a test-specimen, said method comprising:

obtaining a test-specimen from a subject;

adding a buffer; and

reading the results from the test-cassette.

In certain embodiments, the present invention provides and utilizes compositions and materials for conducting a lateral flow assay (e.g., a lateral flow immunoassay). Lateral flow assays are based on the principles of immunochromatography and can be used to detect, quantify, test, measure, and monitor a wide array of analytes, pathogens (e.g., SARS-CoV-2), and the like.

Neutralizing antibodies identified using the disclosed methods can specifically bind to any known or as yet undiscovered coronavirus, such as, for example, coronavirus OC43, coronavirus 229E, coronavirus NL63, coronavirus HKU1, MERS-CoV, SARS-CoV, or SARS-CoV-2 (COVID-19). In some embodiments, the neutralizing antibodies are directed against SARS-CoV-2 (COVID-19). In the context of the present disclosure a “neutralizing antibody” is an antibody that binds to a virus (e.g., a coronavirus) and interferes with the virus' ability to infect a host cell. Coronavirus spike proteins are known to elicit potent neutralizing-antibody and T-cell responses. The ability of a virus (e.g., coronavirus OC43, coronavirus 229E, coronavirus NL63, coronavirus HKU1, MERS-CoV, SARS-CoV, or SARS-CoV-2 (COVID-19)) to gain entry into cells and establish infection is mediated by the interactions of its “viral-ACE2 binding protein” (e.g., Spike glycoproteins, and the like) with human cell surface receptors.

As used herein, the phrase “viral-ACE2 binding protein” refers to any full length protein, functional fragment thereof (e.g., an RBD domain, and the like) that functions to bind to ACE2 (e.g., human ACE2) to facilitate gaining entry into cells to establish a coronavirus infection, e.g., a SARS-Cov-2 infection. Exemplary viral-ACE2 binding proteins are well-known in the art, and include spike proteins (e.g., SARS CoV-2 spike protein) or RBD domains thereof, and the like. In the case of coronaviruses, Spike proteins are large type I transmembrane protein trimers that protrude from the surface of coronavirus virions. Each Spike protein comprises a large ectodomain (comprising S1 and S2), a transmembrane anchor, and a short intracellular tail. The S1 subunit of the ectodomain mediates binding of the virion to host cell-surface receptors through its receptor-binding domain (RBD). The S2 subunit fuses with both host and viral membranes, by undergoing structural changes.

SARS-CoV-2 utilizes the Spike glycoprotein to interact with cellular receptor ACE2 (Zhou et al., Nature 579: 270-273, doi:10.1038/s41586-020-2012-7 (2020); Hoffmann et al., Cell, 50092-8674(0020)30229-30224, doi:10.1016/j.cell.2020.02.052 (2020) doi:10.1016/j.cell.2020.02.052 (2020). The amino acid sequence of the SARS-CoV-2 spike protein has been deposited with the National Center for Biotechnology Information (NCBI) under Accession No. QHD43416. Binding with ACE2 triggers a cascade of cell membrane fusion events for viral entry. The high-resolution structure of SARSCoV-2 RBD bound to the N-terminal peptidase domain of ACE2 has recently been determined, and the overall ACE2-binding mechanism is virtually the same between SARS-CoV-2 and SARS-CoV RBDs, indicating convergent ACE2-binding evolution between these two viruses (Gui et al., CellRes 27, 119-129, doi:10.1038/cr.2016.152 (2017); Song et al., PLoS Pathog 14, e1007236-e1007236, doi:10.1371/journal.ppat.1007236 (2018); Yuan et al., Nat Commun 8, 15092-15092, doi:10.1038/ncomms15092 (2017); and Wan et al., J Virol, JVI.00127-00120, doi:10.1128/JVI.00127-20 (2020)). This suggests that disruption of the RBD and ACE2 interaction, e.g., by neutralizing antibodies, would block SARS-CoV-2 entry into the target cell. Indeed, a few such disruptive agents targeted to ACE2 have been shown to inhibit SARS-CoV infection (Kruse, R. L., F1000Res, 9: 72-72; doi:10.12688/f1000research.22211.2 (2020); and Li et al., Nature 426, 450-454; doi:10.1038/nature02145 (2003)). In addition, neutralizing antibodies directed against coronaviruses (also referred to herein as “coronavirus neutralizing antibodies”) have been identified and isolated (see, e.g., Liu et al., Potent neutralizing antibodies directed to multiple epitopes on SARS-CoV-2 spike. Nature (2020). doi.org/10.1038/s41586-020-2571-7; Rogers et al., Science 15 Jun. 2020:eabc7520; DOI: 10.1126/science.abc7520; Alsoussi et al., J Immunol Jun. 26, 2020, ji2000583; DOI:/doi.org/10.4049/jimmunol.2000583; Kreer et al., Cell, S0092-8674(20)30821-7. 13 Jul. 2020, doi:10.1016/j.cell.2020.06.044; Tai et al., J Virol. 2017 Jan. 1; 91(1): e01651-16; and Niu et al., J Infect Dis. 2018 Oct. 15; 218(8): 1249-1260).

The peptide comprising a receptor binding domain (RBD) of a coronavirus spike protein may be prepared using routine molecular biology techniques, such as those disclosed herein. The nucleic acid and amino acid sequences of RBDs of various coronavirus spike proteins are known in the art (see, e.g., Tai et al., Cell Mol Immunol 17, 613-620 (2020). doi.org/10.1038/s41423-020-0400-4; and Chakraborti et al., Virology Journal volume 2, Article number: 73 (2005); and Chen et al., Biochemical and Biophysical Research Communications, 525(1): 135-140 (2020)). An exemplary RBD domain of a SARS-CoV-2 spike protein comprises the following amino acid sequence:

(SEQ ID NO: 1)

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVL

YNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKI

ADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDI

STEIYQAGSTPCNGVEGFNCYFPLQSYGFPTNGVGYQPYRVVVLSFELLH

APATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDI

ADTTDAVRDPQTLEILDITPCS.

In other particular embodiments, an exemplary sequence used herein for the RBD domain corresponds to amino acids 319-541 of SARS-CoV-2 Spike, set forth as follows:

QRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGC VIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCY FPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF (SEQ ID NO:2). Those skill in the art will recognize that functional fragments of SEQ ID NO:1 and/or SEQ ID NO:2 can also be used in the invention methods and devices.

In particular embodiments, the test-specimen is whole blood, plasma or serum. In another embodiment, the test-specimen can also be obtained from saliva. In certain embodiments, the whole blood, plasma or serum is obtained from a patient either known or suspected of recovering from COVID19 disease; or known to have been vaccinated for SARS-CoV-2. In particular embodiments, the plasma is obtained using anti-coagulants such as heparin, dipotassium EDTA or sodium citrate, and the like.

In particular embodiments, an exemplary sequence used herein for the ACE2 domain corresponds to amino acids 18-615 of the full-length human ACE2, set forth as follows:

(SEQ ID NO: 3)

QSTIEEQAKTFLDKENHEAEDLEYQSSLASWNYNTNITEENVQNMNNAGD

KWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLN

TILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWES

WRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDY

SRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLL

GDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVS

VGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMD

DFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKH

LKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGE

IPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYT

RTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWT

LALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD

Those skill in the art will recognize that functional fragments of SEQ ID NO:3 can also be used in the invention methods and devices.

As used herein the term “tag/anti-tag pair” or vice versa (anti-tag/tag pair) refers to 2 moieties that are known to bind (e.g., non-covalently) to each other. For example, tag/anti-tag pairs can be ligand/receptor pairs; where the anti-tag is the binding partner to the tag. In an embodiment, the ACE2 or functional fragment thereof (referred to herein as ACE2 for simplicity) binds to the nitrocellulose membrane through a tag/anti-tag interaction during the assay. In another embodiment, the ACE2 is bound to the nitrocellulose membrane through a tag/anti-tag interaction prior to the assay, for example during manufacturing of or preparation of the assay. The tag/anti-tag interaction can be a noncovalent interaction, such as a protein-ligand interaction. In an embodiment, the noncovalent protein-ligand interaction is an interaction between biotin and avidin or streptavidin. Biotin is conjugated to ACE2, and avidin or streptavidin is conjugated to the nitrocellulose membrane. The high-affinity interaction between biotin and avidin or streptavidin tethers the biotin-ACE2 conjugate to the streptavidin-conjugated sample pad such that the ACE2 is then available to be bound by the viral ACE2-binding protein from the conjugate pad. Streptavidin is a tetramer and each subunit binds biotin with equal affinity; thus, wild-type streptavidin binds four biotin molecules. For some applications it is useful to generate a strong 1:1 complex of two molecules, i.e., monovalent binding. Monovalent streptavidin is an engineered recombinant form of streptavidin which is still a tetramer but only one of the four binding sites is functional. A streptavidin with exactly two biotin binding sites per tetramer (divalent streptavidin) can be produced by mixing subunits with and without a functional biotin binding site. A streptavidin with exactly three biotin binding sites per tetramer (trivalent streptavidin) can also be produced using the same principle as to produce divalent streptavidins. The streptavidin used in the inventive assay can be wild-type (binding four biotins), or it may be monovalent, divalent, or trivalent. Methods of conjugating biotin and streptavidin to proteins and substrates are known to those of skill in the art and any such methods can be used to conjugate biotin or streptavidin to ACE2, and to conjugate biotin or streptavidin to the sample pad.

In another embodiment, the noncovalent protein-ligand interaction is a Halo interaction. Halo-Tag is a 33 kDa mutagenized haloalkane dehalogenase that forms covalent attachments to substituted chloroalkane linker derivatives (Halo-Ligand). Similarly to the streptavidin-biotin connection, the chloroalkane linker extends 1.4 nm into the hydrophobic core of Halo-Tag. Commercially available Halo-ligand derivatives include the invariant chloroalkane moiety followed by 4 ethylene glycol repeats, and a reactive sulfahydryl, succinimidyl ester, amine, or iodoacetamide group, among many other options. Methods of conjugating biotin and streptavidin to proteins and substrates are known to those of skill in the art and any such methods can be used to conjugate Halo-Tag or Halo-Ligand to ACE2, and to Halo-Tag or Halo-Ligand to the sample pad.

In another embodiment, the noncovalent protein-ligand interaction is a His-tag interaction. The His-tag (also called 6×His-tag) contain six or more consecutive histidine residues. These residues readily coordinate with transition metal ions such as Ni2+ or Co2+ immobilized on beads or a resin. The His-tag is added to the recombinant ACE2 used in the assay, with the beads or resin with immobilized Ni2+ or Co2+ conjugated or otherwise bound to the nitrocellulose membrane. Methods of adding His-tags to proteins and beads or resin with immobilized Ni2+ or Co2+ to substrates are known to those of skill in the art and any such methods can be used to add a His-tag to ACE2, and beads or resin with immobilized Ni2+ or Co2+ to the nitrocellulose membrane. In other embodiments, the noncovalent interaction utilizes a ligand tag that is calmodulin-binding peptide, glutathione, amylose, a c-my tag, or a Flag tag. The ligand tag is attached to the ACE2, and the respective ligand-binding molecule is attached to the nitrocellulose membrane using methods known to those of skill in the art. The ligand tag can also be single-strand DNA (ssDNA) attached to the ACE2, where the complementary ssDNA is immobilized on the nitrocellulose membrane.

In another embodiment, the ACE2 is directly bound to the nitrocellulose membrane via covalent bonding. In an embodiment, the covalent bond is amine-glutaraldehyde-amine, where an amine group on ACE2 is conjugated to an amine group either natively present or introduced on the surface of the membrane. In an embodiment, the covalent bond is amine-NHS (N-hydroxysuccinimide), where NHS ester is used as a covalent linking agent. In an embodiment, the covalent bond is carboxylate-1-ethyl-3-(3-dimethylamonipropyl) carbodiimide (EDC)-amine, where carbodiimide is used to form amide linkage between carboxylates and amines. In other embodiments, the covalent bond is carboxylate-EDC+NETS-amine. In an embodiment, the covalent bond is amine/sulfhydryl-epoxide, where epoxides form covalent bonds with primary amines at mild alkaline pH or with sulfhydryl groups (—SH) in the physiological pH range. In an embodiment, the covalent bond is amine-isothiocyanate, where the reaction of an aromatic amine with thiophosgene (CSCl2) yields isothiocyanate (—NCS), which forms a stable bond with primary amine groups. In another embodiment, the covalent bond is amine-azlactone, where azlactone is used to react with nucleophiles such as amines and thiols at room temperature to form amide bonds. In an embodiment, the covalent bond is amine-p-nitrophenyl ester, where p-nitrophenyl ester is reactive to amines across the slightly basic pH range spanning 7-9 and the ester forms a stable amide bond with proteins. In an embodiment, the covalent bond is amine-tyrosinase (TR)-tyrosine. Tyrosinase is a phenol oxidase that oxidizes phenols into 0-quinone (i.e., 1,2-benzoquinone), which is reactive and undergoes reaction with various nucleophiles such as primary amines. In another embodiment, the covalent bond can be sulfhydryl-maleimide, where maleimide is used to form covalent links with the cysteine residues of proteins. In another embodiment, the covalent bond is reactive hydrogen-benzophenone, where during UV exposure, the benzophenone couples with a protein via reactive hydrogen compounds on the protein. When the benzophenone residues are incorporated onto sample pad, the ACE2 can be immobilized to the surface of the sample pad via exposure to UV light. The particular methods of applying these covalent bonding chemistries to conjugation of proteins is known to those of skill in the art. Multiple covalent bonding chemistries can be used together, including with bifunctional linkers, as known to those of skill in the art. An enormous variety of covalent conjugation chemistries beyond those listed here are known to those of skill in the art. See, for example Kim et al. Biomicrofluidics 7, 041501 (2013), Rusmini et al. Biomacromolecules 8, 1775 (2007), and Hermansson Bioconjugate Techniques, 2nd ed. (Academic Press, San Diego, 2008), all incorporated herein by reference.

The covalent bonding chemistries described above are useful not only for directly conjugating ACE2 to the nitrocellulose membrane, but also for conjugating the respective molecules for noncovalent interactions to ACE2 or to the nitrocellulose membrane, for example for conjugating biotin to ACE2 and/or for conjugating avidin or streptavidin to the nitrocellulose membrane. Additionally, spacers such as polyethylene glycol (PEG) chains can be used together with the linkers for such covalent conjugation (e.g., PEG-NETS) to provide space between the ACE2 and nitrocellulose membrane, and/or ACE2 and biotin, and/or avidin or streptavidin and nitrocellulose membrane. Such spacing can be used to provide the ACE2 with more freedom of movement relative to the nitrocellulose membrane and thus greater opportunity to interact with the viral ACE2-binding protein and/or neutralizing antibodies.

In a particular embodiment, ACE2 is bound to biotin; and the sample pad is bound to streptavidin. In typical embodiments, the viral-ACE2-binding protein is an RBD.

As used herein, the term “label” refers to a moiety, the presence of which can be detected using methods well-known in the art for label-detection. In an embodiment, the viral ACE2-binding protein is coupled to a label such that it can be detected when bound to the ACE2 bound to the nitrocellulose membrane, thus demonstrating a lack of neutralizing antibodies in the sample. In an embodiment, the control protein (for example, an anti-IgG monoclonal antibody) is coupled to a label such that it can be detected when bound to its target on the nitrocellulose membrane (for example, IgG), thus demonstrating that the test is functional and has been performed properly. In an embodiment, the viral ACE2-binding protein and control protein are coupled to different labels. In an embodiment, the label for the viral ACE2-binding protein and/or that for the control protein is detectable by the naked eye. In another embodiment, the label for the viral ACE2-binding protein and/or that for the control protein is detectable by fluorescence. In another embodiment, the label for the viral ACE2-binding protein and/or that for the control protein is detectable by chemiluminescence. Methods for coupling the labels to proteins are known to those of skill in the art.

Labels detectable by the naked eye include metal nanoparticles and nanoshells (e.g., green gold nanoshells; red, orange, or blue gold nanoparticles; copper oxide nanoparticles; silver nanoparticles), gold colloid, platinum colloid, polystyrene latex or natural rubber latex colored with respective pigments such as red and blue. Labels detectable by the naked eye include textile dyes, such as for example, a Direct dye, a Disperse dye, a Dischargeable acid dye, a Kenanthol dye, a Kenamide dye, a Dyacid dye, a Kemtex reactive dye, a Kemtex acid dye, a Kemtex Easidye acid dye, a Remazol dye, a Kemazol dye, a Caledon dye, a Cassulfon dye, an Isolan dye, a Sirius dye, an Imperon dye, a phtalogen dye, a naphtol dye, a Levafix dye, a Procion dye, and an isothiocyanate dye. Examples of textile dyes that can be used to label proteins include, for example, Remazol brilliant blue, Uniblue A, malachite green isothiocyanate, and Orange 16 (Remazol orange). Any label known to those of skill in the art to both be fluorescent and used to label proteins can be used.

Fluorescent labels include any of the Alexa fluor dyes, any of the BODIPY dyes, any of the eFluor dyes, any of the Super Bright dyes, fluorescein or a derivative thereof, eosin or a derivative thereof, tetramethylrhodamine, rhodamine or a derivative thereof, Texas red or a derivative thereof, pyridyloxazole or a derivative thereof, NBD chloride, NBD fluoride, ABD-F, lucifer yellow or a derivative thereof, 8-anilino-1-naphthalenesulfonic acid (8-ANS) or a derivative thereof, Oregon green or a derivative thereof, Pacific blue or a derivative thereof, Pacific green or a derivative thereof, Pacific orange or a derivative thereof Cy3, Cy5, Cyanine7, Cyanine5.5, or coumarin or a derivative thereof. Fluorescent labels include any fluorescent protein, such as green fluorescent protein (GFP), red fluorescent protein (e.g., dsRed), cyan fluorescent protein, blue fluorescent protein, yellow fluorescent protein, enhanced green fluorescent protein (EGFP), or any derivative of such fluorescent proteins thereof. Any label known to those of skill to both be fluorescent and be used to label proteins can be used.

Chemiluminescent labels include enzyme labels that catalyze formation of ATP which is then assayed by the firefly reaction or that catalyze formation of peroxide which is determined by luminol, isoluminol, or peroxyoxalate CL. Such enzyme labels include luciferase and horseradish peroxidase. Any label known to those of skill in the art to both be chemiluminescent and used to label proteins can be used.

In particular embodiments, the label is selected from a nanoparticle, bead, latex bead, aptamer, oligonucleotides, proteins and/or a quantum dot. In another embodiment, the conjugate pad further comprises a mixture of RBD coupled to a nanoparticle and control-antibody coupled to a nanoparticle. In one embodiment, the RBD is coupled to a gold nanoshell (GNS) and the control-antibody is a monoclonal antibody (e.g., a mouse Mab, or the like) coupled to a gold nanosphere (GNP). In particular embodiments, reading the results from the test-cassette further comprises determining the intensity of a test-line in the test-cassette compared with a reference standard.

As used herein, the phrase “reference standard” refers to a control set of values, either obtained simultaneously with the assay results or obtained from a previous control experiment such they they are indicative of the level of NAbs present in the test-specimen (see, e.g., FIG. 2A, FIG. 2B, FIG. 8A, FIG. 8B, FIG. 9A, FIG. 9B, FIG. 11, FIG. 12, or the like). In a particular embodiment, the reference standard is a scorecard.

In certain embodiments, the level of anti-SARS-CoV-2 NAbs in the test-specimen is reported as falling within a range of pre-determined values. As used herein, the phrase “reported as falling within a range of pre-determined values” refers to the manner in which the level of anti-RBD NAbs are analyzed relative to the reference standard or set of control values. The range of pre-determined values can be as few as two levels of NAb values (or concentrations) up top about 10 or more distinct concentration (or quantity) levels of NAbs present in the test-specimen. In one embodiment corresponding to 2 levels of Nab values, for example, falling either above or below a predetermined set value may indicate the presence of sufficient protective anti-RBD NAbs, such that there is a greater likelihood there is protection from getting a subsequent coronavirus infection. In another embodiment, a particular embodiment, the range of pre-determined values corresponds to high, moderate or low/non-neutralizing relative to three respective controls (see FIG. 2A). In another embodiment, the range of pre-determined values corresponds to High (H), Moderate-High (MH), Moderate to Moderate-High (M-MH), Moderate (M), Moderate to Not Detectable (M-ND) and Not Detectable (ND) (see FIG. 2B). Thus, those of skill in the art will appreciated that any number of NAb concentrations and/or quantity levels can be used to identify particular test-specimens being assayed for particular purposes, e.g., those test-specimens above a specified level can be advantageously useful in convalescent therapy

In a particular embodiment, the invention methods are referred to herein as the IMMUNOPASS SARS-Cov-2 Neutralizing Antibody Rapid Test is a lateral flow immunochromatographic assay for semi-quantitative measurement of antibodies that neutralize SARS-CoV-2 in human serum or plasma (see FIG. 1). In a particular embodiment, this test uses immobilized polystreptavidin (test line T) and goat anti-mouse IgG (control line C) on a nitrocellulose strip. In other embodiments, the conjugate pad contains recombinant SARS-CoV-2 antigen (Spike protein RBD domain from SARS-CoV-2) conjugated with dark green gold Nanoshells and a mouse antibody conjugated to red gold Nanospheres. The sample pad contains tagged (e.g., biotinylated) human ACE2 protein.

During testing, in a particular embodiment, anti-RBD antibodies in plasma or serum bind to RBD-conjugated dark green gold Nanoshells in the test cassette. When assay (chase) buffer is added to the sample well, the dried components on the strip interact with plasma or serum from whole blood. If the sample contains antibodies that prevent RBD from binding to ACE2 (neutralizing antibodies), the test will show a light or ghost Test line. If the sample does not contain, or contains low levels of neutralizing antibodies, RBD-gold Nanoshells and ACE2-biotin will interact forming a dark green Test line.

To serve as a procedural control, a colored line should always appear in the control line region, indicating that the proper volume of specimen has been added and membrane wicking has occurred.