Coronavirus Point-of-Care Agglutination Assay

Abstract

The present disclosure provides reagents and methods for the rapid detection of SARS-CoV-2. The assay can be performed in a point-of-care or laboratory setting. The assay of the present disclosure utilizes beads coated with anti-SARS-CoV-2 surface antigen antibodies that undergo agglutination in the presence of SARS-CoV-2. In some embodiments, the antibodies recognize the S1 or S2 spike protein of SARS-CoV-2. In some embodiments, the antibodies recognize the receptor binding domain (RBD) of the S1 spike protein. In some embodiments, the antibodies recognize the N-terminal domain (NTD) of the S1 spike protein. In some embodiments, the antibodies are neutralizing antibodies.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 065,993, filed August 14, 2020, which is incorporated by reference in its entirety. [Background technology]

[0002] background Many assay technologies are available for detecting viral infections in individuals. Various assays for SARS-CoV-2, the causative agent of COVID-19, have been developed and are in development. However, many of these require laboratories, complex equipment, specialized trained personnel, and a significant amount of time to perform the assay. These requirements limit the usefulness of such assays for the rapid identification of infected individuals who could potentially transmit COVID-19 in public settings. Summary of the Invention [Means for solving the problem]

[0003] Abstract There is a need for a point-of-care (POC) assay for the detection and / or quantification of the SARS-CoV-2 virus, the causative agent of COVID-19. Disclosed herein are reagents and methods for a rapid POC assay that can be performed anywhere a potentially infected individual may be encountered, where the assay can be completed in a few minutes (e.g., 5 minutes), and produces a visually detectable result. The assay requires only a saliva sample from the subject being tested.

[0004] In further embodiments, the assay procedure can be adapted for use in clinical laboratories where multiple samples are processed in parallel, e.g., in 96-well plates, to allow for high throughput. In one aspect of these embodiments, the amount of virus in the sample can be quantified.

[0005] The assay of the present disclosure utilizes beads coated with anti-SARS-CoV-2 surface antigen antibodies that undergo agglutination in the presence of SARS-CoV-2. In some embodiments, the antibodies recognize the S1 or S2 spike protein of SARS-CoV-2. In some embodiments, the antibodies recognize the receptor binding domain (RBD) of the S1 spike protein. In some embodiments, the antibodies recognize the N-terminal domain (NTD) of the S1 spike protein. In some embodiments, the antibodies are neutralizing antibodies. In other embodiments, the surface antigen recognized by the antibodies is the hemagglutinin protein, which is the matrix (M) protein of the envelope (E) protein. In some embodiments, the neutralizing antibodies have an IC50 of 3-4 nM by competitive ELISA.

[0006] In some embodiments, the beads are latex beads, in some embodiments, the beads are magnetic beads, in some embodiments, the beads have a deep or intense color.

[0007] In some embodiments, the beads are fluorescent, in some embodiments, the beads are tagged with luciferase or other luminescent agents, and in still other embodiments, the beads are tagged with any other visually or spectrophotometrically detectable signal generating agent.

[0008] In some embodiments, the beads have a diameter of 1.3 to 1.9 μm or about 0.55 μm to about 2.7 μm. In some embodiments, the beads have a diameter of about 550 nm. In some embodiments, the beads have a diameter of about 1.6 μm. In some embodiments, the beads have a diameter of about 2.7 μm. These diameter sizes refer to the diameter of the beads before the addition of streptavidin, antibodies, or other coatings or modifications.

[0009] In some embodiments, the beads are streptavidinated. In some embodiments, the beads are carboxy beads and streptavidin is covalently attached to the beads using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry. In some embodiments, the antibody is biotinylated and coated onto the streptavidin beads via biotin-streptavidin binding. In some embodiments, the antibody coating comprises 25-35 μg of antibody per mg of beads. In some embodiments, the antibody coating comprises 30 μg of antibody per mg of beads.

[0010] In some embodiments, the anti-SARS-CoV-2 antibody is conjugated to the beads with an affinity reagent other than streptavidin. In various embodiments, the conjugated affinity reagent is Protein A, Protein G, an anti-Fc antibody, an anti-species Ig antibody (e.g., goat anti-rabbit Ig or rabbit anti-mouse Ig), or an anti-label antibody (e.g., an antibody that recognizes biotin, fluorescein, dextran, etc.), depending on the nature of the anti-SARS-CoV-2 antibody and what it may be modified with.

[0011] In some embodiments, the antibody is directly coupled to carboxy beads using EDC chemistry, hi some embodiments, the antibody is directly coupled to tosyl-activated beads or to epoxy-activated beads.

[0012] The basic assay procedure involves combining anti-SARS-CoV-2 surface antigen antibody-coated beads with a saliva sample from a subject to be tested for SARS-CoV-2 infection; mixing the beads and the saliva sample to form a mixture; incubating the mixture; and detecting whether agglutination occurs. In some embodiments, the incubation is for about 5 minutes. In some embodiments, the incubation is at room temperature. In other embodiments, the incubation is at 37°C.

[0013] In variations on the basic assay procedure, anti-SARS-CoV-2 surface antigen antibody-coated beads are combined in a multiwell plate with a saliva sample from a subject to be tested for SARS-CoV-2 infection; the beads are incubated at room temperature for 1 to 10 minutes (e.g., 5 minutes); and the plate is shaken on a plate shaker for a period of time. In some embodiments, the period is 5 minutes. In some embodiments, the shaker is at room temperature. In other embodiments, the plate shaker is at 37°C. In some embodiments, a linear shaking motion is used. In some embodiments, the shaking is fast, e.g., about 1000 cycles / minute.

[0014] In some embodiments, the saliva sample is contained in an oral rinse fluid. The oral rinse can be obtained by swishing and gargling with a saline solution (e.g., 0.9% saline solution) and expectorating into a collection container. In some embodiments, 5 mL of saline solution is used for rinsing. In some embodiments, rinsing and gargling are allowed to proceed for 30 seconds.

[0015] Alternatively, in some embodiments, a nasal or nasopharyngeal swab specimen is collected in a saline solution or other transport medium by agitating the swab in the fluid to disperse any viruses present in the specimen. In another variation, the swab is agitated in a mouthwash containing a saliva sample so that viruses present in either can be detected. The transport medium is UTM®, Universal Transport Medium. TM (Copan Diagnostics, Inc. Murrieta, CA), a room-temperature stable viral transport medium (consisting of Hanks' balanced salt solution, bovine serum albumin, L-cysteine, gelatin, sucrose, L-glutamic acid, HEPES buffer, phenol red, sucrose, vancomycin, amphotericin B, and colistin) for the collection, transport, maintenance, and long-term frozen storage of viral and other infectious specimens. The medium is isotonic and nontoxic to mammalian host cells.

[0016] In some embodiments, each individual's saliva sample is collected and processed separately for detection. In some embodiments, the mixture from multiple individuals, or aliquots thereof, is transferred to separate wells of a multi-well plate after mixing or incubating, and detection is performed in a microplate reader or microarray digital reader. In some embodiments, a thickening agent is added to the mixture after incubating but before transferring. In some embodiments, the thickening agent is FICOLL.

[0017] In some embodiments, known amounts of virus (eg, a dilution series) are assayed to generate a calibration curve from which the amount of virus in an individual's saliva sample can be quantified.

[0018] In some embodiments, the assay is qualitative, distinguishing between the presence and absence of virions but not providing a quantification of the number of virions present. In some embodiments, the qualitative assay is capable of detecting at least as many as 100 virions per milliliter. In some embodiments, the qualitative assay is capable of detecting at least as many as 10 virions per milliliter. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 shows assayed saliva samples that were polymerase chain reaction (PCR) positive (left) or PCR negative (right) for SARS-CoV-2. Bead-based agglutination is clearly visible in the vial on the left (virus-containing sample), whereas no bead agglutination is observed in the vial on the right (virus-free sample).

[0020] [Figure 2-1] Figures 2A-D show the change in interference (in nanometers) as an RBD polypeptide binds to and dissociates from an antibody immobilized on a bio-layer interferometry sensor. The vertical dotted line at 0 seconds indicates when the biosensor was immersed in a solution of RBD polypeptide, and the vertical dotted line at 240 seconds indicates when the biosensor was removed from the RBD polypeptide solution and immersed in buffer. Each plot shows three pairs of tracings. Each pair represents the actual data and fitted curve from which rate constants (KD, Ka, and Kd) were derived. For some of the pairs, the data and fitted traces were indistinguishable. In each plot, the top trace is for 100 nM RBD polypeptide, the middle trace is for 10 nM RBD polypeptide, and the bottom trace is for 0 nM RBD polypeptide. 2A - Ty1; 2B - MM57; 2C - R001; and 2D - MM43. [Figure 2-2]Figures 2A-D show the change in interference (in nanometers) as an RBD polypeptide binds to and dissociates from an antibody immobilized on a bio-layer interferometry sensor. The vertical dotted line at 0 seconds indicates when the biosensor was immersed in a solution of RBD polypeptide, and the vertical dotted line at 240 seconds indicates when the biosensor was removed from the RBD polypeptide solution and immersed in buffer. Each plot shows three pairs of tracings. Each pair represents the actual data and fitted curve from which rate constants (KD, Ka, and Kd) were derived. For some of the pairs, the data and fitted traces were indistinguishable. In each plot, the top trace is for 100 nM RBD polypeptide, the middle trace is for 10 nM RBD polypeptide, and the bottom trace is for 0 nM RBD polypeptide. 2A - Ty1; 2B - MM57; 2C - R001; and 2D - MM43.

[0021] [Figure 3] Figure 3 shows images of the agglutination reaction of Ty1-coated 550 nm beads incubated with SARS-CoV-2 negative (top) and positive (bottom) samples from Example 5.

[0022] [Figure 4] Figure 4 shows images of the agglutination reaction of R001-coated 2.7 μm beads incubated with SARS-CoV-2 negative (top) and positive (bottom) samples from Example 5.

[0023] [Figure 5] Figure 5 shows images of the agglutination reaction described in Example 6. From left to right are five images: beads only, Figure 7 negative sample + 100 μL of beads, 100 μL positive sample + 50 μL of beads, 100 μL positive sample + 100 μL of beads, and 100 μL positive sample + 200 μL of beads.

[0024] [Figure 6] 6A-B show images of the agglutination reaction described in Example 7. An array of four wells can be seen. The top well contains a negative sample and the bottom well contains a positive sample. The left pair of wells holds an agglutination reaction using a 2:1 ratio of beads:sample, and the right pair of wells holds an agglutination reaction using a 1:1 ratio of beads:sample. 6A shows the raw image, and 6B shows the same image after processing.

[0025] [Figure 7] Figure 7 shows images from a bead dilution agglutination assay described in Example 8. Dilutions proceed from left to right in ratios of 100:75:50:25:10:0. The top row received negative samples and the bottom row received positive samples.

[0026] [Figure 8] Figure 8 is a schematic of a point-of-care device for performing a SARS-CoV-2 agglutination assay.

[0027] [Figure 9] Figure 9A-B. A test card for a smartphone-based point-of-care SARS-CoV-2 agglutination assay is shown in 9A. The test card demonstrates proper positioning of the test chamber and provides an appropriate background for image capture. 9B shows a mock-up results screen from the image-reading smartphone app.

[0028] [Figure 10-1]Figures 10A-C depict SARS-CoV-2 assay results. 10A reports the object sum area (OSA) from saliva samples from infected and uninfected individuals, titered virions, and saline, as well as the signal-to-noise ratio (S / N) for each positive sample relative to the average of three negative samples at the 15-minute endpoint and for the slope from 0 to 3 minutes. 10B is a plot of the virion count vs. clump count reading over 0 to 3 minutes. 10C is a plot of the slope of the virion count vs. clump reading at 15 minutes. 10B-C plot only the three negative samples (open triangles) and the virion serial dilutions (filled circles). [Figure 10-2] Figures 10A-C depict SARS-CoV-2 assay results. 10A reports the object sum area (OSA) from saliva samples from infected and uninfected individuals, titered virions, and saline, as well as the signal-to-noise ratio (S / N) for each positive sample relative to the average of three negative samples at the 15-minute endpoint and for the slope from 0 to 3 minutes. 10B is a plot of the virion count vs. clump count reading over 0 to 3 minutes. 10C is a plot of the slope of the virion count vs. clump reading at 15 minutes. 10B-C plot only the three negative samples (open triangles) and the virion serial dilutions (filled circles).

[0029] [Figure 11] FIG. 11 shows an overview of one embodiment of the complete agglutination testing process.

[0030] [Figure 12] FIG. 12 shows a saliva device for collecting saline mouthwash to be tested in one particular embodiment.

[0031] [Figure 13]Figures 13A-B show results from the agglutination assay interpreted by the slope of OSA change. 13A plots OSA over time for each of the samples tested, labeled with the sample number. 13B shows images of each sample. See Table 4 for sample numbers and descriptions. DETAILED DESCRIPTION OF THE INVENTION

[0032] explanation One simple technique for detecting or quantifying multivalent analytes (e.g., virus particles) is agglutination. Agglutination typically involves clumping particles together, relying on antibody-antigen binding. Classic examples of agglutination-based assays include ABO blood typing and the monospot assay for Epstein-Barr virus infection. Agglutination can occur when the antibody component of the reaction can form bridges between particles bearing two (or more) antigens, with each antigen-bearing particle being bridged to multiple other particles. If there are too many antibodies to an antigen, the antibodies will saturate all of the binding sites on the antigen-bearing particles, so in fact, no cross-linking occurs and no agglutination occurs. Steric factors can also interfere with agglutination, for example, when the binding geometry is such that an antibody bound to a particle bearing one antigen cannot reach a particle bearing a second antigen, or when the binding kinetics are such that both binding sites of an antibody (e.g., assuming a bivalent antibody) bind to a particle bearing the same antigen. Steric factors such as these can often be overcome by attaching antibodies to beads, effectively increasing their valency, reach and location diversity.

[0033] Binding the antibodies to beads also addresses an additional challenge: virus particles and antibody molecules are so small that even if agglutination does not occur, aggregates (clusters) may not be visually observable. By binding the antibodies to visible beads, successful agglutination, at least in the case of agglutination, can be detected with the naked eye (or a camera).

[0034] This agglutination method detects virions, not RNA or antigens dissociated from the virions. Agglutination occurs best with intact virion samples (e.g., fresh saliva, saline rinse, or swab / saline rinse samples, as well as gamma-irradiated saliva-based samples). Heat inactivation can denature or destroy the virion structure of some virions, which reduces the efficiency of agglutination of damaged virions or virion fragments. Because agglutination relies on substantially intact virions, it is less prone to false-positive tests resulting from residual antigens and RNA after the infection has resolved. This contrasts with ELISA- and PCR-based tests, which detect antigens and RNA, respectively, whether or not associated with intact virions.

[0035] In certain embodiments, anti-SARS-CoV-2 surface antigen antibodies are biotinylated and reacted with streptavidin-labeled magnetic beads. Some of these features are convenient but not essential. The biotin-avidin reaction is widely used to attach antibodies to beads and other substrates; the reaction is well understood and the necessary reagents are readily available. Nevertheless, other chemistries for attaching antibodies to beads are known and can be utilized. Similarly, while magnetic beads can be easy to process, the general assay protocol disclosed herein does not utilize magnetism. However, magnetic beads typically contain iron and have a dark brown color that facilitates visual detection. Non-magnetic beads can also be used, but should be of a dark and / or intense color that facilitates visual detection. In addition to brown, black and dark shades of blue, green, red, and purple are suitable, whereas white, yellow, and tan, for example, are less preferred. Latex beads are often used in agglutination assays and are available in a variety of colors.

[0036] In certain embodiments, the anti-SARS-CoV-2 surface antigen antibody is a neutralizing antibody that binds to the receptor-binding domain of the SARS-CoV-2 spike protein. The neutralizing activity of the antibody is not essential for the assay. Any antibody that binds to a multivalent site on the virus particle is potentially useful. However, the receptor-binding domain is an accessible and well-conserved site, making it well suited for this purpose. Monoclonal antibodies that recognize the SARS-CoV-2 S1 and S2 spike proteins and receptor-binding domain (including neutralizing monoclonal antibodies derived from mice and rabbits) are commercially available from several sources. Neutralizing single-domain antibody fragments derived from alpacas have also been generated (Hanke et al., bioRxiv 2020.06.02.130161, which is incorporated herein by reference in its entirety). Although these antibody fragments (nanobodies) are monovalent, they are nevertheless suitable for use in agglutination assays when bound to beads. This is because the beads are multivalent.

[0037] Alpaca nanobodies (Ty1) often offer several advantages. They are enzymatically biotinylated at the C-terminus using Sortase A, which ensures consistent orientation of the nanobody relative to the bead, with its antigen-binding site facing away from the bead (optimal for virus binding). This contrasts with randomly biotinylated antibodies, which are bound to the bead in various orientations (some of which may be sterically hindered). Ty1 can also recognize the RBD in both its "up" and "down" conformations (also referred to as "open" and "closed" conformations, respectively). The small size of the nanobody (only 12.5 kD) allows it to bind to the RBD of each of the three S1 promoters in a single spike protein. The small size of the nanobody also means that there are more virus-binding sites per mg of antibody coating a bead than with a full-sized antibody.

[0038] Antibody affinity is also an important parameter to consider when selecting an antibody for use as an agglutination reagent, with higher affinity being associated with tighter agglutination. In some embodiments, the antibody has a KD of greater than 2.5, 5.0, 7.5, 9.0, or 9.5, as measured in phosphate-buffered saline (PBS) by biolayer interferometry. In various embodiments, the KD is in the range of 2.5, 5.0, 7.5, 9.0, or 9.5 to 10. In some embodiments, the KD is about 2.8. In some embodiments, the KD is about 9.9.

[0039] In certain embodiments, magnetic beads with free carboxylate groups (carboxybeads) are covalently coupled to streptavidin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry, blocked, and stripped to prevent nonspecific binding. A biotinylated antibody that recognizes SARS-CoV-2 is then attached using biotin-streptavidin binding. Procedures and variations of this process are known in the art (see, e.g., PCT / US2020 / 039503, which is incorporated herein by reference for all that it teaches about making and using streptavidin-coated beads to bind biotinylated molecules). It is desirable that both the streptavidin-coated beads and the subsequent biotin-antibody-coated streptavidin beads be monodisperse to maximize binding surface area, and that the assay is based on bead aggregation / aggregation. Vigorous bead mixing, shear mixing, and sonication can be used to ensure the beads are mixed, uniform, and monodisperse. Monodisperse beads (unaggregated beads) also provide optimal control results with PCR-negative saliva samples because the beads should not aggregate or clump in the absence of their viral target.

[0040] In some embodiments, antibodies are directly coupled to carboxy beads using EDC chemistry. In some embodiments, antibodies are directly coupled to tosyl-activated beads or to epoxy-activated beads. In still other embodiments, the antibodies are non-covalently coated onto beads using coordination chemistry.

[0041] Embodiments utilizing streptavidin are described throughout this disclosure. However, additional embodiments including alternatives (e.g., avidin, deglycosylated avidin (neutravidin), CaptAvidin, monomeric avidin) are also contemplated. Natural and recombinant versions of streptavidin, as well as alternatives thereof, are also contemplated. These reagents may be referred to as means for attaching biotin.

[0042] Instead of streptavidin or its analogs, other affinity reagents can be bound to beads and used for conjugating anti-SARS-CoV-2 antibodies. In some embodiments, the conjugated affinity reagent is an anti-biotin antibody and can conjugate biotinylated anti-SARS-CoV-2 antibodies. In some embodiments, the conjugated affinity reagent recognizes a label such as fluorescein, dextran, or His tag and can conjugate fluoresceinated, dextran-modified, His-tagged, or otherwise labeled anti-SARS-CoV-2 antibodies, respectively. In some embodiments, the conjugated affinity reagent can recognize the Fc region of an antibody (e.g., Protein A, Protein G, or anti-Fc antibody) and can conjugate anti-SARS-CoV-2 antibodies that include an Fc region. In some embodiments, the conjugated affinity reagent is an antibody that recognizes a species-specific epitope in an immunoglobulin (Ig), such as a goat anti-rabbit Ig antibody or a rabbit anti-mouse Ig antibody, and can conjugate an anti-SARS-CoV-2 antibody that is a rabbit or mouse antibody, respectively.

[0043] Beads of various sizes can be used. In some embodiments, the beads have a diameter of about 500 nm to about 2.7 μm, or about 1.3 to about 1.9 μm. In some embodiments, the beads have a diameter of 1.6 μm. In some embodiments, the beads have a diameter of 2.7 μm. Bead size can affect sedimentation time, with larger beads settling faster. In a flat-bottom 96-well plate containing 100-200 μL of liquid (without thickener), sedimentation of 2.7 μm beads can be complete in about 3 minutes, and sedimentation of 1.6 μm beads can be complete in about 5 minutes. 550 nm beads can take longer to sediment. However, as beads aggregate / aggregate, they can form very large clumps, which can decrease the sedimentation time of the aggregates compared to monodisperse beads.

[0044] The beads can be coated with various amounts of antibody. In some embodiments, the beads contain about 10 to about 50 μg of antibody per mg of beads, or about 25 to about 35 μg of antibody per mg of beads. In some embodiments, the beads contain 30 μg of antibody per mg of beads.

[0045] In some embodiments, to obtain a saliva sample for assay, the oral cavity is rinsed with saline solution and the rinse is collected (rinsed and spat out) to obtain a mouthwash. In some embodiments, 5 mL of saline solution is vigorously rinsed and gargled for 30 seconds, then spat into a collection container (e.g., a collection tube with a funnel) (OralDNA Labs' Universal Mouthwash Collection Kit is suitable for this purpose; FIG. 12).

[0046] However, in some embodiments, the obtained saliva sample is centrifuged to remove cellular material.

[0047] To perform the assay, a suspension of beads is added to a saliva sample. In some embodiments, the saliva sample is included in a mouthwash. In some embodiments, the components are combined using a 1 mg / mL suspension of anti-SARS-CoV-2 antibody-coated beads in a 1:2 bead:saliva sample ratio. In aspects of these embodiments, the concentration of the bead suspension can be greater than or less than 1 mg / mL depending on the desired total reaction volume and dilution factor due to the saliva sample. In some embodiments, 0.5 mL of bead suspension is added to 1 mL of saliva sample (e.g., mouthwash). Other embodiments use other bead:sample ratios and bead concentrations (see Examples 6 and 8). The combined bead suspension and saliva sample are mixed and then incubated. In some embodiments, the incubation is performed at room temperature. In other embodiments, the incubation is performed at 37°C. In some embodiments, the incubation is for at least 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes, 5 to 15 minutes, 2 minutes, 3 minutes, or 5 minutes. In some embodiments, the incubation for aggregation and precipitation occurs in one step.

[0048] In general, it is important to maintain an optimized ratio of beads (agglutination reagent) to saliva sample volume. This can be adjusted for larger or smaller size reactions (e.g., reactions performed in glass vials versus microtiter plates). However, detection sensitivity can be increased by using a larger volume of saliva sample (and proportionally more agglutination reagent) because there is a larger mass of material from which to form agglutinates.

[0049] In an alternative embodiment, when magnetic beads are used, the beads are magnetically separated from the suspending fluid and then resuspended in the saliva sample (mouthwash). The final concentration of beads can be similar to that described above (e.g., about 0.33 mg beads / mL). In various embodiments, the bead concentration in the agglutination reaction can be 0.1-1.5 mg / mL, e.g., 0.13 mg / mL, 0.33 mg / mL, 0.5 mg / mL, 0.67 mg / mL, 0.75 mg / mL, 1.0 mg / mL, or 1.33 mg / mL, or any range bounded by a pair of these values. The resuspended beads are then incubated at room temperature or 37°C in some embodiments. In some embodiments, the incubation is carried out for at least 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes, 5-15 minutes, 2 minutes, 3 minutes, or 5 minutes, by which time agglutination has occurred.

[0050] In further embodiments, there is no explicit mixing step prior to incubation other than that resulting from the addition of the components. In further embodiments, the static incubation is followed by incubation on a shaker (e.g., a plate shaker). In some embodiments, the shaking incubation is performed at room temperature, while in other embodiments, it is performed at an elevated temperature, e.g., 37°C. In some of these embodiments, the shaking incubation is preceded by a static incubation, e.g., for about 1 minute or about 5 minutes. In other embodiments, there is no explicit static incubation after the dispensing of the reagents into the plate is complete.

[0051] This agglutination is a visible event and can be read by a variety of methods, ranging from visual assessment by a human to fully automated high-throughput plate imagers. In many embodiments, this is sufficient to visually assess the formation of aggregates (with the naked eye). In alternative embodiments, aggregates can be detected with a camera (e.g., a smartphone camera), an optical density reader, a spectrophotometer, a luminometer, a fluorimeter, or a digital flow cell, a particle sizer (e.g., Anton Paar Litesizer 500). All of these modes of measuring aggregates can be referred to as processes for detecting agglutination.

[0052] For higher throughput applications, an aliquot of the agglutination reaction (i.e., the bead and saliva sample mixture) can be transferred to a well of a multi-well plate (e.g., a 96-well plate) after mixing or incubation. Detection of aggregate formation can be achieved, for example, with a microplate reader (by optical density) or with a microarray digital reader (by image analysis). These methods of detecting aggregates can also be referred to as a process for detecting agglutination or a high-throughput process for detecting agglutination. In such an embodiment, it is also possible to include a dilution series of a known virus sample as a calibration curve so that the amount of virus present in the saliva sample can be quantified.

[0053] Beads and aggregates tend to settle together. In some embodiments, this may be undesirable. To inhibit settling, a thickening agent can be added to the agglutination reaction with mixing after the incubation step and before the detection step, and before the transfer step, if used. One suitable thickening agent is FICOLL (a neutral, highly branched, high-mass hydrophilic polysaccharide).

[0054] Microarray digital readers are camera-based and use image analysis. They have a narrow focal depth. This contrasts with microplate readers, which operate by passing a beam of light through the entire depth of the sample and measuring optical density, conductance, or absorbance (although some readers take measurements at multiple spots in the sample). As a result of the narrow focal depth, microarray digital readers can, and often do, only see the bottom of the wells. In such cases, sedimentation of aggregates is desirable. However, it is also possible to change the focal plane throughout the entire depth of the sample, scan, and build a three-dimensional image. This process typically takes slightly more than one minute per plate. When scanning through multiple focal planes, the use of a thickening agent to inhibit the settling of beads and aggregates may be desirable. A more uniform distribution of beads and aggregates may also be advantageous when using a microplate reader, so a thickening agent may also be used in such embodiments.

[0055] Some microplate readers scan across wells to generate two-dimensional (total area of ​​interest) or three-dimensional profiles from which the surface area or volume of detected peaks and troughs can be calculated and used to quantify agglutination. In some embodiments, the assay results are based on readings (e.g., volume, surface area, or total area of ​​interest) at a specific time point after the start of the assay (e.g., 2-20 minutes, or any integer value therein (e.g., 15 minutes)). In some embodiments, the assay results are based on the rate of change (slope) of the readings over a time interval from zero to 2-20 minutes, or any integer value therein (e.g., 0-3 minutes).

[0056] Thus, the assays described herein can be used to detect SARS-CoV-2 infection for any purpose. However, they are well suited to environments where speed and / or simplicity are advantageous or required. For example, these assays can be used for home screening before returning to work after SARS-CoV-2 infection, or for screening to identify individuals before allowing them to enter any public gathering place (such as a school, government office, place of worship, store, sporting event, airport, or air travel).

[0057] When using magnetic beads, a confirmatory PCR test of a positive result can be performed on the assayed sample itself. Reagents that extract viral RNA are added to the well or vial, the beads are magnetically separated, and the fluid is transferred to a PCR reaction. A confirmatory PCR test can also be used to confirm that the virus detected is indeed SARS-CoV-2 and not a cross-reacting coronavirus, or to identify which strain of SARS-CoV-2 is present.

[0058] Alternatively, the agglutination reagent can be used to purify saliva samples prior to PCR testing if the presence or potential presence of interfering substances in the saliva is an issue. The anti-SARS-CoV-2 antibody-coated magnetic beads are added to the saliva sample and incubated to bind the virus. The beads (agglutinated or unagglutinated) are magnetically separated and washed. Viral RNA is then extracted as usual, the beads are again magnetically separated, and the viral RNA-containing fluid is collected for PCR analysis. [Example]

[0059] Example The following non-limiting examples are provided for illustrative purposes only to facilitate a more complete understanding of the representative embodiments contemplated herein, and should not be construed as limiting any of the embodiments described herein.

[0060] Example 1 Bead preparation Magnetic beads with diameters of 550 nm and 1.6 μm and with free carboxyl groups were coated with streptavidin, which was covalently attached as follows: 1. 1.6 μm and 550 nm carboxy beads were coated with streptavidin using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) chemistry. 2. 40 mM MES (pH 5.2) was the coupling buffer. 3. 0.5 mg of streptavidin was used per 1.0 mg of beads during coupling. 4. 0.1 mg of EDC was added per 1.0 mg of beads (18.6 mol EDC per mol COOH on 1.6 μm beads; 9 mol EDC per mol COOH on 550 nm beads). The EDC was added to a 40 mM MES solution (pH 5.2) at 10 mg / mL. 5. The above reaction was carried out at a bead concentration of 10 mg / mL. 6. The EDC reaction was allowed to mix at room temperature for approximately 12 hours. 7. The beads were removed from passively adsorbed streptavidin to ensure covalent attachment and blocked with a polymer blocker to reduce non-specific binding by the beads and ensure monodispersity. 8. The beads were washed in TBS with Tween® 20 and NaN3 (10 mM Tris, 150 mM NaCl, 0.05% Tween® 20, 0.05% NaN3) at a bead concentration of 10.0 mg / mL.

[0061] Three anti-SARS-CoV2 neutralizing antibodies, which recognize the receptor-binding domain of the SARS-CoV2 spike protein, were biotinylated as follows: 1. The three antibodies were brought to 1 mg / mL (100 μg of antibody) in PBS (40 mM NaHPO, 10 mM KHPO, 137 mM NaCl, 3 mM KCl) pH 8.0 in a total volume of 100 μL. a. Sino Biological catalog number 40591-MM43 lot HB14AP2001, 100 μg, 1.63 mg / mL in PBS (comp. unk.). As reported by the manufacturer, typical IC of 1.41 μg / mL by neutralization assay and 0.857 nM by ELISA. 50 A mouse monoclonal antibody having b. Sino Biological catalog number 40592-MM57 lot HB14AP2002, 100 μg, 1.98 mg / mL in PBS (composition unknown). As reported by the manufacturer, typical IC of 0.41 μg / mL by neutralization assay and 3.694 nM by ELISA. 50 A mouse monoclonal antibody having c. Sino Biological catalog number 40592-R001 lot HA14MY2101, 100 μg, 5.36 mg / mL in PBS (composition unknown). Typical IC of 0.11 μg / mL by neutralization assay and 0.59 nM by ELISA, as reported by the manufacturer. 50 A rabbit monoclonal antibody having 2. QUANTA Biotin-dPEG4-TFP Ester Catalog No. 10009 Lot AF1-A0402-011 was reconstituted in DMSO (LT Baker #9224-01 Lot 0000217025) and added to the antibody solution. 3. A 10x molar excess of biotin linker over antibody was used. 4. Incubation time was 2 hours at RT on a rotary mixer. 5. The biotinylated antibody was not desalted after biotinylation. 6. The above biotinylated antibodies were coated onto streptavidin-coated beads of three different sizes: a. 550nm - MM43 antibody was coated at 50µg antibody per mg of beads. b. 1.6 μm-MM57 antibody was coated at 30 μg antibody per mg of beads. c. 2.7 μm R001 antibody was coated at 10 μg antibody per mg of beads. The 550 nm and 1.6 μm beads were prepared as described above. The 2.7 μm bed was purchased streptavidinylated (Agilent PN 6727-1003; 2220 pmoles / mg bead binding capacity (biotin-4-fluorescein binding capacity)) and was removed and blocked as described above. 7. The beads were coated at a concentration of 10 mg / mL. 8. The incubation time for the antibodies and beads was 2 hours at room temperature on a rotary mixer. 9. The beads were washed three times (by magnetic separation) in TBS (10 mM Tris, 150 mM NaCl, 0.05% Tween® 20, 0.05% NaN3) with TWEEN® 20 and NaN3 at a bead concentration of 1.0 mg / mL.

[0062] Example 2 agglomeration A 0.5 mL suspension of 1 mg / mL 1.6 μm diameter streptavidin-conjugated magnetic beads coated with 30 μg / mg MM57 antibody was added to 1 mL each of SARS-CoV-2 PCR-positive and -negative mouthwashes. The combined reagents were mixed and incubated for 5 minutes at room temperature. Agglutinated beads were visually evident in virus-positive samples but not in virus-negative samples (Figure 1).

[0063] Example 3 Determination of antibody dissociation constants Biolayer interferometry (BLI) was used to determine the binding affinity of four antibodies to the RBD portion of the SARS-CoV-2 S1 spike protein, as described above: MM57, R001, MM43, and Ty1 (an alpaca-derived nanobody). MM57, R001, and MM43 were biotinylated at random lysines, while Ty1 was biotinylated at a unique C-terminal site to ensure uniform orientation relative to the substrate to which it was bound.

[0064] BLI is an optical analysis technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized proteins on a biosensor chip and an internal reference layer. In this case, the biosensor chip was coated with streptavidin, and the antibody to be tested was then bound to the streptavidin to form the immobilized protein layer. The biosensor chip was immersed in 100 nM, 10 nM, or 0 nM solutions of RBD polypeptide in phosphate-buffered saline (PBS), and the change in interference was observed as the RBD polypeptide bound to the antibody. When saturation was reached, the biosensor chip was immersed in buffer (PBS), and the change in interference was observed as the antibody dissociated (Figure 2A-D). From these data, the KD, Ka, and Kd were calculated (Table 1). [Table 1]

[0065] Example 4 SARS-CoV-2 aggregation on nanobody-coated beads This experiment was performed using three different beads: streptavidin-coated JSR Magnosphere TM MS55 / Carboxyl, 550 nm beads (bead size 1); JSR Magnosphere coated with streptavidin TMAssays were performed with Ty1 monobiotinylated nanobodies coated onto MS160 / Carboxyl, 1.6 μm beads (bead size 2); and Agilent 2.7 μm LodeStars streptavidin beads (bead size 3). The beads were treated as described above and suspended at 1 mg / ml in Tris-buffered saline (TBS; 10 mM Tris, 150 mM NaCl, 0.05% TWEEN® 20, 0.05% NaN3, pH 7.4).

[0066] The positive and negative samples were mouthwash + inserted nasal swab. Virus was heat-inactivated by incubation at 60°C for 10 minutes. This partially denatured viral proteins and was expected to somewhat reduce the sensitivity of the assay compared to fresh patient samples. Positive and negative samples were validated by PCR. The detection limit in the PCR assay was approximately 15 virions / mL, corresponding to a cycle number (Ct) of approximately 38 cycles. Negative samples had a Ct > 40. The positive sample used in this experiment had a Ct of 27.9.

[0067] 100 μL of positive or negative sample was combined with 50 μL, 100 μL, or 200 μL of bead suspension for each of the three sizes in individual vials. After 5 minutes of incubation at room temperature, agglutination was scored visually on a scale of 0 - no agglutination, 1 - slight agglutination, 2 - good agglutination, and 3 - excellent agglutination. The results are shown in Table 2. [Table 2]

[0068] The vials were randomized and presented to each of six laboratory technicians who were asked to score them for the presence or absence of brown precipitate. All six easily selected nine positive and three negative samples with 100% positive agreement and 100% negative agreement.

[0069] Example 5 SARS-CoV-2 agglutination with various antibodies and bead sizes This experiment compared each of the three bead sizes, as described in Example 4, coated with each of the four antibodies characterized in Example 3. The experiment was performed in a 96-well plate. The 96-well plate was pretreated by incubating with 0.023% PLURONIC® F108 and 0.05% TWEEN® 20 in TBS for 5 minutes to block virus or bead binding to the well surface, after which the blocking solution was aspirated.

[0070] Then, 66 μL of the various bead suspensions (1 mg / mL in TBS) were dispensed into the wells of a black, clear, flat-bottom 96-well plate. 33 μL of sample (0.9% saline mouthwash + inserted nasal swab, heat inactivated) was added to the beads, and the plate was gently swirled manually to mix. The positive sample used in this experiment had a Ct of 27.9. The beads were incubated at room temperature and allowed to settle (>5 minutes). The wells were then imaged with a microarray reader focused on the bottom of the well. [Table 3]

[0071] In this experiment, the Ty1 nanobody showed the least sensitivity to bead size. The MM43 antibody, which had one of the lower affinities, consistently performed poorly. Images from Ty1-coated 550 nm beads and R001-coated 2.7 μm beads incubated with SARS-CoV-2 negative and positive samples are shown in Figures 3 and 4, respectively.

[0072] Example 6 Aggregation at different bead-to-sample ratios This experiment was performed in a similar manner to Example 5 above, using a blocked 96-well plate with Ty-1 coated 1.6 μm beads. The positive sample was heat inactivated and had a PCT Ct of 27.9. Five samples were prepared: beads alone; 100 μL negative mouthwash + 100 μL bead suspension (1 mg / mL); and 100 μL positive mouthwash + 50 μL, 100 μL, or 200 μL bead suspension (sample to bead (volume:volume) ratios of 2:1, 1:1, and 1:2). The wells were imaged using a microarray reader by setting the focal plane at the bottom of the well and taking an image after the beads had completely settled. The image is shown in Figure 5.

[0073] Example 7 Flocculation with an additional shaking step This experiment was performed using Ty1-coated 2.7 μm beads in a blocked 96-well plate. Positive (Ct of 27.9) and negative samples were mouthwash plus inserted nasal swabs. One set of reactions used 66 μL of beads (1 mg / mL in TBS) and 33 μL of sample (2:1 ratio), while the second set used 50 μL of each (1:1 ratio). The reactions were incubated for approximately 1 minute and then placed on a plate shaker at 37°C for 5 minutes. The shaker was set to linear shaking at approximately 1000 cycles per minute. The plate was removed and photographed from above using a smartphone camera equipped with an external 15x magnification lens while the plate was illuminated from below. The aggregates form a dark band near the center of the well. Image processing can be used to further highlight the differences between positive and negative results. See Figures 6A and 6B.

[0074] Example 8 Titration of bead concentration in agglutination reactions This experiment was performed in a blocked 96-well plate using R001-coated 2.7 μm beads. The positive and negative samples were mouthwash + inserted swab, with the positive sample having a PCR Ct of 25.92. A six-concentration bead dilution series with a bead content ratio of 100:75:50:25:10:0 was used. The well with the highest concentration of beads received 66 μL of a 2 mg / mL suspension in TBS, and each successive well in the dilution series received a proportionally smaller volume. The missing volume was made up of a diluent of 0.9% saline (the same as the mouthwash). Each well received diluent, 99 μL of sample, and beads, in that order. The reactions were set up in a blocked, clear, flat-bottom, black 96-well plate as described above. After adding the reagents, the plate was allowed to sit for 5 minutes without mixing and then placed on a plate shaker at 37°C for 5 minutes. The shaker was set to linear shaking at approximately 1000 cycles / min. The plate was then removed from the plate shaker and images were acquired with a smartphone (see Figure 7). At the highest bead concentration, there is relatively little difference between the positive and negative samples, but as the bead concentration is reduced, increasingly clear bands form in the center of the positive wells, while in the negative wells the beads remain in a diffuse and somewhat circular pattern.

[0075] Example 9 Point-of-care device for SARS-CoV-2 agglutination assay A point-of-care device that minimizes sample handling and manipulation is depicted in FIG.

[0076] Mouthwash (or similar sample) is added to a saline rinse reservoir and the plunger is depressed, mixing the sample and collecting it in a loading chamber for reading. No other manipulation of the sample or assay reagents is required. Agglutination is assessed visually or with a smartphone camera and app.

[0077] The device includes a cylinder and a microfluidic module. The cylinder contains a plunger and includes three spaces and four channels. The internal volume of the cylinder above the plunger head constitutes the saline wash reservoir, in which the sample is received. The internal volume of the cylinder below the plunger head is divided into two by a deformable plastic or Mylar® film. One of the spaces constitutes the bead chamber, which contains a suspension of anti-RBD antibody-coated beads. The other space is the saline measure chamber, whose volume is smaller than the volume of the saline reservoir and the expected volume of the sample. A channel (depicted on the left side of the cylinder) allows sample to drain from the saline wash reservoir into the saline measure chamber, and a vent channel (not depicted) allows air to escape. Filling the saline measure chamber provides a fixed volume of sample without the user having to measure or transfer the sample. Channels from each of the chambers lead to the fluid mixing path of the microfluidic module. The two channels may optionally communicate in an in-line mixer (not depicted) before connecting to the fluid mixing path. The read-fill chamber may be fitted with an overfill valve to allow air to escape as the chamber fills but prevent liquid from exiting the chamber. The top of the read-fill chamber is transparent (e.g., thin, clear optical polystyrene) so that the presence or absence of aggregate formation can be observed and / or photographed. The bottom of the read-fill chamber may be transparent like the top. In that case, the device should be read (photographed) on a light-colored background. Alternatively, the bottom may be white-colored opaque plastic.

[0078] Once enough sample has been expelled to fill the saline measurement chamber, the plunger can be depressed. Depressing the plunger breaks the seals holding the bead suspension and sample in their chambers, and the two fluids flow through the channel at the bottom of the cylinder, through a T-junction, an in-line mixer (if present), and a fluid mixing path to the read-fill chamber. Mixing can also be aided by a twist in the fluid mixing path. The device can be adjusted to operate with 100 μL each of bead suspension and sample.

[0079] Example 10 POC - Smartphone App Based Testing The smartphone camera is used to image the completed agglutination reaction, and the results are read using an app. Diagnostic results are based on image analysis by AI trained on positive and negative samples. This technology eliminates human error in sample reading, making fast, easy, and accurate testing accessible to everyone.

[0080] To perform this POC test, use the following steps:

[0081] 1) Remove the plastic spit-out cup from the kit. 2) Remove the mouthwash bottle from the kit, remove the twist cap, and place the entire 5 mL of mouthwash into your mouth, swirl your mouth for 25 seconds, and gargle for 5 seconds. 3) Spit the 5 mL mouthwash saliva sample into a plastic spit-out cup. 4) Take the capillary lancet device (saliva sample collection device) from the kit and insert it into the mouthwash saliva sample in the spit-out cup and draw 50 uL of saliva sample into the capillary lancet. 5) Remove the agglutination reagent sealed vial from the kit and mix (invert) 10 times to mix the beads into a suspension (if using a clear vial, the user may be instructed to mix until a light brown color appears with no dark spots or clumps of beads visible). 6) When inserting the capillary lancet with the saliva sample into the agglutination reagent, insert the capillary lancet into the agglutination reagent by puncturing the seal until the capillary lancet fits snugly or tightly into the agglutination reagent vial, forming a seal. 7) The saliva sample and the agglutination reagent are mixed by inverting 10 times. 8) Remove the cap covering the dropper end of the capillary lancet device inserted into the agglutination reagent vial. 9) Place the test card with the test chamber on a flat surface, or equivalently, place the test chamber onto the test card so that it covers the outline or square of the test chamber. 10) Hold the dropper end over the test chamber and gently crush the bottle to add enough drops to cover the bottom of the test chamber. 11) The app has a timer that instructs you on when and how to take a photo of the sample in the test chamber in front of the test card (Figure 9A). 12) The test results are displayed on the app (Figure 9B).

[0082] Test results displayed on a smartphone screen may include a time code indicating when the individual tested negative (or positive) (see Figure 9B, left panel). The negative test code may serve as a badge to facilitate entry into public settings (e.g., airplane travel, work, sporting events, churches, schools, etc.). Instead of or in addition to the time code, a barcode or QR code may be present. The resulting screen may use distinct colors for either the text and images or the background. For example, a negative result screen may use green or blue, a positive test screen may use red, orange, or yellow; and an inconclusive test screen may use blue or black. Other color schemes using different colors for each result are also possible.

[0083] Example 11 Confirmatory testing of aggregated viruses It is conceivable that there may be coronaviruses that cross-react with any particular anti-SARS-CoV-2 surface antigen antibody, potentially resulting in false-positive results. Furthermore, multiple strains of SARS-CoV-2 have been identified, and which strain infects an individual may be of clinical or epidemiological importance. Therefore, it may be useful to confirm that the virions captured in the aggregates are in fact SARS-CoV-2 and / or to identify which strain is present. Both goals may be achieved by subjecting the captured virions to PCR (or similar) testing.

[0084] The aggregate-bound virions are heat-inactivated and lysed, the beads are magnetically separated, and the liquid containing the lysed virus is aspirated and subjected to PCR as usual. Alternatively, the virions are eluted by washing with a glycine (pH 2.5) elution buffer. The solution is then adjusted to a neutral pH, the beads are magnetically separated, and the eluted purified virions are aspirated and subjected to PCR as usual.

[0085] Example 12 Sample cleaning with anti-SARS-CoV-2 surface antigen antibody-coated magnetic beads Saliva is not necessarily pure and may contain substances from coffee, chewing gum, tobacco, food, etc. that can interfere with PCR assays. Anti-SARS-CoV-2 surface antigen antibody-coated magnetic beads provide a method to remove interference from saliva samples that would interfere with PCR testing. For this application, it is not necessary for agglutination to occur, as long as virions bind to the beads.

[0086] Anti-SARS-CoV-2 surface antigen antibody-coated magnetic beads are added to a saliva sample to capture SARS-CoV-2 virions for magnetic washing with 0.9% saline to wash away these interferences. The bead-bound virions are then heat-inactivated and lysed, the beads are magnetically separated, and the liquid containing the lysed virus is aspirated and subjected to PCR as usual.

[0087] Alternatively, the virions are eluted by washing with glycine (pH 2.5) elution buffer. The solution is then adjusted to neutral pH, the beads are magnetically separated, and the eluted purified virions are aspirated and subjected to PCR as usual.

[0088] Example 13 Model Protocol for SARS-CoV-2 Assembly / Agglutination Assay Summary of Method: Paramagnetic microparticles (PMPs) coated with antibodies against the SARS-CoV-2 spike RDB protein are introduced into a human sample derived from a nasal swab mixed with saline. The PMPs react by clumping together. The clumped PMPs are then imaged and counted, for example, with a BioTek Cytation 5 microscope cell counter. Clumps are counted within a size gate. The counted clumps or the rate of clump growth over time is proportional to the presence of SARS-CoV-2 virions in the sample.

[0089] material: 1) Streptavidin-coated PMPs (1.6 μm diameter) conjugated with biotinylated MM57 anti-RBD monoclonal antibody (see Example 1). 2) Nasal collection swab (Copan PN502CS01 Copan Diagnostics Inc, 26055 Jefferson Ave, Murrieta, CA 92562). 3) Saline solution (0.09% NaCl in purified water). 4) Cytation 5 Cell Imaging Multi-Mode Reader. 5) Alpaqua Catalyst TM 96, 96 well Slotted Ring Magnet Plate SKU: A000550 (Alpaqua Inc. 100 Cummings Center, Suite #424A, Beverly, MA 01915). 6) Reaction plate (Corning PN353910 Corning Inc, 1 River Front Plaza Corning NY 14831). 7) Half-area reading plate (Greiner PN675090 Greiner BioOne GMBH Maybach St 2, 72636 Frickenhausen, Germany). 8) TTA: Tris-buffered saline with 0.05% Tween® 20 and 0.05% sodium azide. The protocol can be adapted to use equivalent reagents from other suppliers.

[0090] Protocol 3 Sample Collection 1) Give a sterile sample swab to the patient. 2) Instruct the patient to gently swab the anterior nasal cavity by rotating the swab against the nasal cavity five times. 3) Place the swab directly into 3 mL of saline in a 15 mL collection tube. 4) Cap the tube and vortex. 5) Remove the liquid into a secondary collection tube and centrifuge. 6) The clear centrifuged liquid is removed for examination.

[0091] Testing Protocol 1) Add 50 μL of centrifuged (21380 rcf for 10 minutes) patient sample to the reaction plate. 2) Add 50 μL antibody-conjugated PMP at 1 mg / mL in TTA to each 50 μL patient sample in the reaction plate. 3) Place on a vibrating shaker (500 RPM) at 30°C for 30 minutes. 4) Place the reaction plate on a 96-place magnet for 5 minutes. 5) Condition the reading plate by adding 15 μL TTA to each well. 6) Resuspend the pellet in the reaction plate using pipette mixing. 7) Add 10 μL of resuspended PMPs from step 6 to the half area read plate. 8) Read: Place plate into Cytation 5 and read kinetically at 3 minute intervals for 15 minutes. 9) Export the following OSA size gate data: a. 2 to 30 microns (preferred) b. 15-20 microns c. 25-30 microns d. Or calculate the slope from the data measurements at 0 and 3 minutes 10) Use saline signal +15% as the positive cutoff. 11) Positive samples have a signal > saline + 15%.

[0092] Example 14 SARS-CoV-2 Assay Results Positive and negative patient saliva samples and serially diluted SARS-CoV-2 virus were assayed essentially as described in Example 13 (above). Data collection gates were set at 2-30 microns, and readings were taken in 3-minute increments from 0 to 15 minutes (Figure 10A). Saliva samples from four SARS-CoV-2-infected patients with PCT assay results were obtained from Access Genetics (Eden Prairie, MN). SARS-associated coronavirus 2 (USA-WA1 / 2020 isolate, gamma-irradiated (BEI Resources, Manassas, VA)) was used to generate 10 virions of SARS-CoV-2 in saline. 6 ~10 2 Ten-fold serial dilutions of virions / mL were prepared.

[0093] The OSA readings (two-dimensional projections of beads) for three negative samples (one saline sample and two saliva samples from uninfected volunteers) were averaged, and the cutoff between positive and negative samples was set at 115% of the mean OSA reading for the negative samples, with a 15-minute endpoint (665,667) and a slope from 0 to 3 minutes (137,900). The signal-to-noise ratio (S / N; Figure 10A) between the mean OSA readings for each positive and negative sample was also determined, demonstrating that positive and negative samples could be easily distinguished. The OSA readings for each of the samples were plotted both at the 15-minute endpoint (Figure 10B) and the slope from 0 to 3 minutes (Figure 10C). (Negative samples were arbitrarily plotted as 0.1 virions / mL, since there is no true zero on the logarithmic scale.) Again, positive and negative samples could be easily distinguished. The results of the assay are qualitative, not quantitative. There was no correlation between virion concentration (or PCR cycle number) and read magnitude.

[0094] Example 15 SARS-CoV-2 Assay Results (Nasal Swab) The assay was performed under conditions modified from those described in the previous example. Nasal swab samples, RBD-coated latex beads, and titrated virion samples from SARS-CoV-2 infected and uninfected individuals (as determined by PCR) were assayed. 20 μL of nasal swab sample was added to 50 μL in a 96-well plate. 80 μL of PMP conjugated with an anti-RBD monoclonal antibody was then added to each well and incubated at 37°C on an external plate heater for 5 minutes. The plate was inserted into a Biotek Cytation 5 plate reader at ambient temperature, and OSA readings were collected within a 9-99 micron particle size window (Figures 13A-B). The slope was calculated over a 17-21 minute interval.

[0095] As seen in Table 4 (below), a slope of less than about 1000 correlated with a negative sample. 10 virions per sample was below detection by PCR but gave a positive signal in the agglutination assay. [Table 4] *A negative PCR result is interpreted as a Ct ≥ 40. **PCR-confirmed patient samples from Access Genentics

[0096] Finally, while aspects of the present specification have been emphasized by reference to specific embodiments, it should be understood that those skilled in the art will readily recognize that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Accordingly, it should be understood that the disclosed subject matter is in no way limited to the particular methods, protocols, and / or reagents, etc., described herein. Accordingly, various modifications or variations of the disclosed subject matter or alternative configurations can be made in accordance with the teachings herein without departing from the spirit of the specification. Finally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Therefore, the present invention is not limited to that precisely as shown and described.

[0097] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect such variations to be utilized by those skilled in the art, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of all possible variations of the above-described embodiments is encompassed by the present invention unless otherwise indicated herein or clearly contradicted by context.

[0098] The grouping of alternative embodiments, elements, or steps of the invention should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is deemed to include the group as modified and thus fulfill all Markush group descriptions used in the appended claims.

[0099] Unless otherwise specified, all numbers expressing properties, items, quantities, parameters, characteristics, terms, etc. used in the specification and claims should be understood to be modified in all instances by the term "about." As used herein, the term "about" means that the so-qualified property, item, quantity, parameter, characteristic, or term encompasses a range of 10% above and below the value of the stated property, item, quantity, parameter, characteristic, or term. Thus, unless indicated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical designation should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values ​​setting forth the broad scope of the invention are approximations, the numerical ranges and values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical range or value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of numerical ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each individual value of the numerical range is incorporated herein as if it were individually recited herein.

[0100] As used in the context of describing the present invention (particularly in the context of the claims below), the terms "a," "an," "the," and similar referents should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better clarify the invention and does not limit the scope of the invention as claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0101] Certain embodiments disclosed herein may be further limited in the claims using the language "consisting of" or "consisting essentially of." When used in a claim, the transitional term "consisting of" excludes any element, step, or ingredient not specified in the claim, whether added at the time of filing or by amendment. The transitional term "consisting essentially of" limits the scope of the claim to specific materials or steps, as well as those that do not materially affect the basic and novel characteristics. Embodiments of the invention so claimed are essentially or explicitly described and enabled herein.

[0102] All patents, patent publications, and other publications referenced and identified herein are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that may be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents. The present invention provides, for example, the following items. (Item 1) 1. A method for detecting SARS-CoV-2 infection in one or more individuals, the method comprising: a) individually combining anti-SARS-CoV-2 surface antigen antibody-coated beads with a saliva sample from each of said one or more individuals to form a mixture corresponding to each individual; b) mixing the beads and saliva sample for each individual mixture; c) incubating the mixture; and d) detecting whether agglutination occurs in the mixture of individuals; The method includes: (Item 2) 2. The method of claim 1, wherein the surface antigen is an S1 or S2 spike protein. (Item 3) 3. The method of claim 2, wherein the antibody recognizes the receptor binding domain or N-terminal domain (NTD) of the S1 spike protein. (Item 4) 4. The method according to any one of items 1 to 3, wherein the saliva sample is contained in a mouthwash. (Item 5) 5. The method of claim 4, wherein the mouthwash is obtained by having each of the one or more individuals rinse and gargle with a saline solution in the individual's oral cavity and then expectorate into a collection container. (Item 6) 6. The method according to item 4 or 5, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads are colored and have a deep or intense hue. (Item 7) 7. The method of claim 6, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads are magnetic. (Item 8) 8. The method according to any one of items 1 to 7, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads are streptavidin-conjugated. (Item 9) 9. The method of claim 8, wherein the antibody is biotinylated and the biotin moiety is bound to streptavidin. (Item 10) 10. The method according to any one of items 1 to 9, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads have a diameter of 1.3 to 1.9 μm. (Item 11) Item 11. The method according to item 10, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads have a diameter of 1.6 μm or more. (Item 12) 10. The method according to any one of items 1 to 9, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads have a diameter of 2.7 μm. (Item 13) 13. The method according to any one of items 1 to 12, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads contain 25 to 35 μg of antibody per 1 mg of beads. (Item 14) 14. The method of claim 13, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads contain 30 μg of antibody per 1 mg of beads. (Item 15) 15. The method according to any one of items 1 to 14, wherein the incubating step is carried out at room temperature. (Item 16) 15. The method according to any one of items 1 to 14, wherein the incubating step is carried out at 37°C. (Item 17) 17. The method according to any one of items 1 to 16, wherein each mixture is incubated for 2 to 5 minutes. (Item 18) 18. The method according to any one of items 1 to 17, wherein the detecting step comprises visual observation. (Item 19) 18. The method according to any one of items 1 to 17, wherein the detecting step comprises the use of a camera. (Item 20) 18. The method according to any one of items 1 to 17, wherein the detecting step comprises the use of an optical density meter. (Item 21) 18. The method of any one of items 1 to 17, comprising transferring an aliquot from each individual mixture to a well of a multiwell plate after the mixing or incubating step, and detecting whether agglutination has occurred for each individual mixture. (Item 22) 22. The method of claim 21, wherein the detecting step comprises using a microplate reader. (Item 23) 22. The method of claim 21, wherein the detecting step comprises using a microarray digital reader. (Item 24) 24. The method of claim 23, further comprising adding a thickening agent to the mixture after the incubating step but before the transferring step. (Item 25) 25. The method of claim 24, wherein the thickening agent is FICOLL. (Item 26) a) combining samples containing known amounts of virions with anti-SARS-CoV-2 surface antigen antibody-coated beads to form a standard curve mixture corresponding to each amount of virion; b) mixing the beads and virion samples for each standard curve mixture; c) incubating the standard curve mixture; and d) after said mixing or incubating step, transferring each standard curve mixture to a well of said multi-well plate and detecting whether agglutination has occurred for each standard curve mixture; 26. The method of any one of items 21 to 25, further comprising: wherein the standard curve mixture serves as a calibration curve by which the amount of virus in the saliva sample can be quantified. (Item 27) 1. A method for detecting SARS-CoV-2 infection in one or more individuals, the method comprising: detecting whether agglutination occurs in the incubated mixture corresponding to each individual; wherein each mixture comprises anti-SARS-CoV-2 surface antigen antibody-coated beads and a saliva sample from one individual. (Item 28) Item 28. The method according to Item 27, wherein the incubated mixture is incubated at room temperature for 2 to 5 minutes. (Item 29) 28. The method according to item 27, wherein the incubated mixture is simultaneously shaken for 1 to 5 minutes. (Item 30) 30. The method of claim 29, wherein the incubation is carried out at 37°C. (Item 31) 31. The method according to any one of items 1 to 30, wherein the anti-SARS-CoV-2 surface antigen antibody is an alpaca-derived nanobody. (Item 32) 32. The method of claim 31, wherein the nanobody is Ty1.

Claims

1. 1. A method for detecting the presence of intact SARS-CoV-2 virions in one or more individuals, the method comprising: a) individually combining anti-SARS-CoV-2 surface antigen antibody-coated beads with a saliva sample from each of said one or more individuals to form a mixture corresponding to each individual; b) mixing the beads and saliva sample for each individual mixture; c) incubating the mixture; and d) detecting whether agglutination occurs in the mixture of individuals; The method includes:

2. 2. The method of claim 1, wherein the surface antigen is an S1 or S2 spike protein.

3. 3. The method of claim 2, wherein the antibody recognizes the receptor binding domain or N-terminal domain (NTD) of the S1 spike protein.

4. The method of any one of claims 1 to 3, wherein the saliva sample is contained in a mouthwash.

5. 5. The method of claim 4, wherein the mouthwash is obtained by having each of the one or more individuals rinse and gargle with a saline solution in the individual's oral cavity and then expectorate into a collection container.

6. The method of claim 4 or 5, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads are colored and have a deep or intense color.

7. 7. The method of claim 6, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads are magnetic.

8. The method according to any one of claims 1 to 7, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads are streptavidin-conjugated.

9. The method of claim 8 , wherein the antibody is biotinylated and the biotin moiety is bound to streptavidin.

10. The method of any one of claims 1 to 9, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads have a diameter of 1.3 to 1.9 μm.

11. 11. The method of claim 10, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads have a diameter of 1.6 μm or less.

12. The method of any one of claims 1 to 9, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads have a diameter of 2.7 μm.

13. The method of any one of claims 1 to 12, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads contain 25 to 35 μg of antibody per mg of beads.

14. 14. The method of claim 13, wherein the anti-SARS-CoV-2 surface antigen antibody-coated beads contain 30 μg of antibody per mg of beads.

15. The method of any one of claims 1 to 14, wherein the incubating step is carried out at room temperature.

16. The method of any one of claims 1 to 14, wherein the incubating step is carried out at 37°C.

17. The method of any one of claims 1 to 16, wherein each mixture is incubated for 2 to 5 minutes.

18. The method of any one of claims 1 to 17, wherein the detecting step comprises visual observation.

19. The method of any one of claims 1 to 17, wherein the detecting step comprises the use of a camera.

20. The method of any one of claims 1 to 17, wherein the detecting step comprises the use of an optical density meter.

21. 18. The method of any one of claims 1 to 17, comprising transferring an aliquot from each individual mixture to a well of a multi-well plate after the mixing or incubating step, and detecting whether agglutination has occurred for each individual mixture.

22. 22. The method of claim 21, wherein the detecting step comprises the use of a microplate reader.

23. 22. The method of claim 21, wherein the detecting step comprises the use of a microarray digital reader.

24. 24. The method of claim 23, further comprising adding a thickening agent to the mixture after the incubating step but before the transferring step.

25. 25. The method of claim 24, wherein the viscosity increasing agent is FICOLL.

26. a) combining samples containing known amounts of virions with anti-SARS-CoV-2 surface antigen antibody-coated beads to form a standard curve mixture corresponding to each amount of virion; b) mixing the beads and virion samples for each standard curve mixture; c) incubating the standard curve mixture; and d) after said mixing or incubating step, transferring each standard curve mixture to a well of said multi-well plate and detecting whether agglutination has occurred for each standard curve mixture; wherein the standard curve mixture serves as a calibration curve by which the amount of virus in the saliva sample can be quantified.

27. 1. A method for visually detecting the presence of intact SARS-CoV-2 virions in one or more individuals, the method comprising: detecting whether agglutination occurs in the incubated mixture corresponding to each individual; wherein each mixture comprises anti-SARS-CoV-2 surface antigen antibody-coated beads and a heat-inactivated saliva sample from one individual, said beads having a diameter of 0.55 μm to about 2.7 μm.

28. 28. The method of claim 27, wherein the incubated mixture is incubated at room temperature for 2 to 5 minutes.

29. 28. The method of claim 27, wherein the incubated mixture is simultaneously shaken for 1 to 5 minutes.

30. 30. The method of claim 29, wherein the incubation is carried out at 37°C.

31. The method of any one of claims 1 to 30, wherein the anti-SARS-CoV-2 surface antigen antibody is an alpaca-derived nanobody.

32. 32. The method of claim 31 , wherein the nanobody is Ty1.

Images