Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54386—Analytical elements
  • G01N33/54387—Immunochromatographic test strips
  • G01N33/54388—Immunochromatographic test strips based on lateral flow
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/08—RNA viruses
  • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2469/00—Immunoassays for the detection of microorganisms
  • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

• the present technology is related to devices, methods, and kits for diagnosing a subject with SARS-CoV-2 vims (“COVID-19”) infection.
• RT-PCR reverse transcriptase polymerase chain reaction
• Other tests include antigen tests, which are currently less accurate such that a “positive” from a current antigen test is often followed by a RT-PCR test. Both tests are inconvenient and expose both the person being tested and the person taking the nasal swab sample to possible infection. Therefore, there is a strong need for a low- cost, accurate test that can be performed by the person being tested in private and that provides the results quickly.
• the present disclosure generally provides a lateral flow immunochromatographic assay (LFA) device comprising one or more combinations of antibodies selected from the group consisting of: a) Exonbio 3E6 detection antibody or a detection antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Exonbio 3E6; Meridian 9547 detection antibody or a detection antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Meridian 9547; and Meridian 9548 capture antibody or a capture antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Meridian 9548; b) Meridian 9548 detection antibody or a detection antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Meridian 9548; Exonbio 3E6 capture antibody or a capture antibody that binds the same antigen,
• the device is a strip, cassette, or cartridge.
• the device comprises a sample application area, a conjugate release area, and a reaction area.
• the conjugate release area comprises one or more detection antibodies selected from the group consisting of: a) Sinobiological 40143-MM08 or a detection antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Sinobiological 40143-MM08; b) Exonbio 3E6 or a detection antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Exonbio 3E6; c) Exonbio 3H1 or a detection antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Exonbio 3H1; d) Exonbio 5B1 or a detection antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Exonbio 5B1
• the reaction area comprises a test line comprising one or more capture antibodies selected from the group consisting of: a) Sinobiological 40143-MM08 or a capture antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Sinobiological 40143-MM08; b) Exonbio 3E6 or a capture antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 3E6; c) Exonbio 3H1 or a capture antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 3H1; d) Exonbio 5B1 or a capture antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 5B 1
• the reaction area comprises a control line comprising one or more control antibodies that bind the detection antibody.
• control antibody is an anti-human immunoglobulin G (IgG) antibody.
• IgG immunoglobulin G
• a concentration of the particles conjugated with the capture antibody set is in the range of approximately 20 to 30 optical density units.
• the present technology provides a method of detecting SARS-CoV-2 infection in a subject comprising obtaining a biological sample from the subject, applying the sample to the device of the above and/or below mentioned embodiments, applying running buffer to the device, and detecting a signal from a capture antibody conjugate, wherein a signal from a capture antibody conjugate indicates that the subject has a SARS-CoV-2 infection.
• the biological sample is saliva.
• the biological sample is from a nasal cavity of the subject.
• the present technology provides a kit comprising one or more combinations of antibodies selected from the group consisting of: a) Exonbio 3E6 detection antibody or a detection antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Exonbio 3E6; Meridian 9547 detection antibody or a detection antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Meridian 9547; and Meridian 9548 capture antibody or a capture antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Meridian 9548; b) Meridian 9548 detection antibody or a detection antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Meridian 9548; Exonbio 3E6 capture antibody or a capture antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and
• the kit further comprises a lateral flow immunochromatographic assay (LFA) device.
• LFA lateral flow immunochromatographic assay
• the one or more combinations of antibodies are packaged separately from the LFA device.
• the one or more combinations of antibodies are pre-loaded onto the device.
• the device comprises a sample application area, a conjugate release area, and a reaction area.
• the reaction area comprises a test line.
• capture antibodies from the one or more combinations of antibodies are pre-loaded onto the test line.
• detection antibodies from the one or more combinations of antibodies are pre-loaded onto the conjugate release area.
• the kit further comprises a detection particle.
• the detection particle is packaged separately from the one or more combinations of antibodies.
• the detection particle is pre-conjugated to a detection antibody from the one or more combinations of antibodies.
• the present technology provides a lateral flow immunochromatographic assay (LFA) device comprising an antibody combination consisting of a detection antibody pair and a capture antibody pair.
• the detection antibody pair consists of: a) Exonbio 3E6 detection antibody or a detection antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Exonbio 3E6, and b) Meridian 9547 detection antibody or a detection antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Meridian 9547.
• the capture antibody pair consists of: a) Sinobiological 40143-MM08 capture antibody or a capture antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Sinobiological 40143-MM08, and b) Meridian 9548 capture antibody or a capture antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Meridian 9548.
• the detection antibody pair is coupled to nanoparticles and pre- loaded at a conjugate release area of the LFA device.
• the capture antibody pair is pre-loaded at a test line of the LFA device.
• the present technology provides a lateral flow immunochromatographic assay (LFA) device comprising an antibody combination consisting of a detection antibody pair and a capture antibody.
• the detection antibody pair consists of: a) Exonbio 3E6 detection antibody or a detection antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Exonbio 3E6, and b) Meridian 9547 detection antibody or a detection antibody that binds the same antigen, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Meridian 9547.
• the capture antibody is Meridian 9548 or an antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Meridian 9548.
• the detection antibody pair is coupled to nanoparticles and pre-loaded at a conjugate release area of the LFA device.
• the capture antibody is pre-loaded at a test line of the LFA device.
• the present technology provides a matched antibody combination for detecting the presence of SARS-CoV-2 vims comprising the antibodies of any of Combination Nos. 1-13 as set forth in the following table:
• Figure 1 illustrates standard curves of antibody pairs evaluated with an enzyme- linked immunosorbent assay (ELISA).
• ELISA enzyme- linked immunosorbent assay
• Figure 2 illustrates antibody pairs in two antibody orientations selected for feasibility testing with a lateral flow assay (LFA).
• LFA lateral flow assay
• Figure 3 illustrates an image of an LFA test strip loaded with the antibody pairs of
• Figure 4 illustrates standard curves of the antibody pairs of Figure 2 evaluated with the LFA.
• Figure 5 illustrates standard curves of a subset of the antibody pairs of Figure 2 evaluated with the LFA.
• Figure 6 illustrates an image of an LFA test strip loaded with antibody pairs under varying pH conditions.
• Figure 7 illustrates images of LFA test strips loaded with antibody pairs under varying EDC/Sulfo-NHS ratios.
• Figure 8 illustrates an image of an LFA test strip loaded with antibody pairs.
• Figure 9 illustrates images of LFA test strips loaded with antibody pairs for testing protein titration with a SARS-CoV-2 nucleocapsid protein recombinant antigen (recombinant N protein).
• Figure 10 is a graph of signal intensities at the test line (TL) of the LFA test strips of Figure 9.
• Figure 11 illustrates an image of an LFA test strip loaded with antibody pairs.
• Figure 12 illustrates an image of an LFA test strip loaded with an antibody pair and tested with various lysis buffers.
• Figure 13 illustrates an image of an LFA test strip loaded with an antibody pair and tested with various amounts of the antibody pair and different buffers.
• Figure 14 illustrates an image of an LFA test strip loaded with the antibody pair of Figure 13 and tested with various amounts of the antibody pair and different buffers.
• Figure 15 illustrates images of LFA test strips loaded with antibody pairs for evaluating detection of recombinant N protein from human saliva samples.
• Figure 16 illustrates images of LFA test strips loaded with an antibody pair for evaluating detection of various strains of COVID-19.
• Figure 17 is a graph of signal intensities at the TL of the LFA test strips of Figure 16.
• Figure 18 illustrates images of LFA test strips loaded with antibody pairs for evaluating detection of recombinant N protein from HEK 293 cells and Escherichia coli ( E . coli).
• Figure 19 illustrates an image of an LFA test strip loaded with antibody pairs for evaluating the limit of detection for a strain of COVID-19.
• Figure 20 illustrates images of LFA test strips loaded with antibody pairs for evaluating detection of recombinant N protein from human nasal swab samples.
• Figure 21 illustrates images of LFA cassettes loaded with antibody pairs for evaluating detection of recombinant N protein from human nasal swab samples.
• antigen refers to any substance capable of eliciting a specific immune response, and may specifically mean any molecule or group of molecules recognizable by at least one antibody.
• the antigen contains at least one epitope (a specific biochemical unit that can be recognized by the antibody).
• antibody refers to both intact immunoglobulin molecules and antigen binding fragments thereof, including but not limited to Fab, Fab’, F(ab’)2, Fv, scFv, or diabody fragments.
• an antibody may be monoclonal or polyclonal, and may fall into any immunoglobulin class (e.g., IgG, IgA, IgM) or subclass.
• the present technology is directed to compositions, methods, and kits for diagnosing a subject with SARS-CoV-2 vims.
• LFAs lateral flow immunochromatographic assays
• Select antibody pairs were evaluated for use in lateral flow assay (LFAs, aka lateral flow immunochromatographic assays) by testing the antibodies on LFA test strips under varying conditions with regard to pH concentration, antibody loading, buffer ratio, recombinant N protein concentration, lysis buffer, amount of conjugated particle, and sample volume.
• Select antibody pairs were further evaluated for detecting recombinant N protein from various strains of COVID-19, human saliva samples, and human nasal swab samples using LFA test strips.
• the limit of detection (LoD) of recombinant N protein and attenuated virus were also determined using the LFA test strips.
• LFA strips in cassettes were also evaluated in comparison to the LFA test strips not in cassettes.
• compositions, devices, and kits comprising one or more antibodies or antibody pairs as disclosed herein, and methods of using these compositions, devices, and kits to diagnose SARS-CoV-2 infection.
• the compositions, devices, kits, and methods provided herein facilitate fast, low-cost, reliable SARS-CoV-2 diagnosis that can be performed by the subject being tested at home, in other public or private settings (e.g., schools, airports, and restaurants), or, by a health care worker for example, in a point of care setting such as a laboratory, doctor’s office, or clinic.
• compositions, devices, and kits provided herein comprise one or antibodies selected from:
• the one or more antibodies may be incorporated into a fusion protein.
• the antibodies are paired as set forth in Table 1.
• the antibodies in Set I are capture antibodies and the antibodies in Set II are detection antibodies.
• the antibodies in Set I are detection antibodies and the antibodies in Set II are capture antibodies.
• compositions comprising two or more of the antibodies disclosed herein.
• the compositions comprise a capture antibody set and a detection antibody set.
• the capture antibody set includes one or more capture antibodies, which may include any of the antibodies in Antibody Set I or Antibody Set II, or capture antibodies that bind the same antigen and/or share the same CDR sequences and/or light and heavy chain variable sequences as any of the antibodies in Antibody Set I or Antibody Set II.
• the detection antibody set includes one or more detection antibodies, which may include any of the antibodies in Antibody Set I or Antibody Set II, or detection antibodies that bind the same antigen and/or share the same CDR sequences and/or variable heavy and light chain variable sequences as any of the antibodies in Antibody Set I or Antibody Set II.
• the compositions provided herein comprise Meridian 9548, Exonbio 3E6, and Meridian 9547.
• the composition may include Meridian 9548 as the capture antibody and Exonbio 3E6 and Meridian 9547 as the detection antibodies.
• the composition may include Meridian 9548 as the detection antibody and Exonbio 3E6 and Meridian 9547 as the capture antibodies.
• the composition may include antibodies that bind the same antigen as Meridian 9548, Exonbio 3E6, and Meridian 9547, and/or which comprise the same CDR and/or variable heavy and light chain sequences as Meridian 9548, Exonbio 3E6, and Meridian 9547.
• the compositions provided herein comprise Sinobiological 40143-MM08, Meridian 9548, Exonbio 3E6, and Meridian 9547.
• the composition may include Sinobiological 40143-MM08 and Meridian 9548 as the capture antibodies and Exonbio 3E6 and Meridian 9547 as the detection antibodies.
• the composition may include Sinobiological 40143-MM08 and Meridian 9548 as the detection antibodies and Exonbio 3E6 and Meridian 9547 as the capture antibodies.
• the composition may include antibodies that bind the same antigen as Sinobiological 40143-MM08, Meridian 9548, Exonbio 3E6, and Meridian 9547, and/or which comprise the same CDR and/or variable heavy and light chain sequences as Sinobiological 40143-MM08, Meridian 9548, Exonbio 3E6, and Meridian 9547.
• the compositions provided herein comprise Exonbio 3E6, Sinobiological 40143-MM08, and Meridian 9548.
• the composition may include Exonbio 3E6 as the capture antibody and Sinobiological 40143-MM08 and Meridian 9548 as the detection antibodies.
• the composition may include Exonbio 3E6 as the detection antibody and Sinobiological 40143-MM08 and Meridian 9548 as the capture antibodies.
• the composition may include antibodies that bind the same antigen as Exonbio 3E6, Sinobiological 40143-MM08, and Meridian 9548, and/or which comprise the same CDR and/or variable heavy and light chain sequences as Exonbio 3E6, Sinobiological 40143-MM08, and Meridian 9548.
• the compositions provided herein comprise Exonbio 3E6 and Sinobiological 40143-MM08.
• the composition may include Exonbio 3E6 as the capture antibody and Sinobiological 40143-MM08 as the detection antibody.
• the composition may include Exonbio 3E6 as the detection antibody and Sinobiological 40143-MM08 as the capture antibody.
• the composition may include antibodies that bind the same antigen as Exonbio 3E6 and Sinobiological 40143-MM08, and/or which comprise the same CDR and/or variable heavy and light chain sequences as Exonbio 3E6 and Sinobiological 40143-MM08.
• the compositions provided herein comprise Sinobiological 40143-MM08, Exonbio 3E6, and Meridian 9547.
• the composition may include Sinobiological 40143-MM08 as the capture antibody and Exonbio 3E6 and Meridian 9547 as the detection antibodies.
• the composition may include Sinobiological 40143-MM08 as the detection antibody and Exonbio 3E6 and Meridian 9547 as the capture antibodies.
• the composition may include antibodies that bind the same antigen as Sinobiological 40143-MM08, Exonbio 3E6, and Meridian 9547, and/or which comprise the same CDR and/or variable heavy and light chain sequences as Sinobiological 40143-MM08, Exonbio 3E6, and Meridian 9547.
• devices for use in LFAs comprising two or more of antibodies provided herein.
• these devices comprise a composition provided herein.
• the devices provided herein comprise one or more capture antibodies and one or more detection antibodies provided herein, including any of the antibodies of Antibody Set I or Antibody Set II, or capture or detection antibodies that bind the same antigen or share the same CDR sequences and/or light and heavy chain variable sequences as any of the antibodies of Antibody Set I or Antibody Set II.
• the devices provided herein comprise strips, cartridges, cassettes, or any other suitable substrate for use in an LFA.
• strips, cartridges, cassettes, or other LFA substrates comprise (a) a sample application area, (b) a conjugate release area, and (c) a reaction area.
• a biological sample to be evaluated is loaded onto the sample application area, then flows across the conjugate release area and the reaction area.
• the device is a dual line device comprising a test line and a control line in the reaction area.
• the sample application area may comprise a sample pad comprising, but not limited to, woven mesh or cellulose fiber.
• the conjugate release area may comprise a conjugate pad comprising, but not limited to, non-woven glass fiber.
• the devices provided herein comprise one or more detection antibodies disclosed herein, including any of the antibodies in Antibody Set I or Antibody Set II, or detection antibodies that bind the same antigen and/or share the same CDR sequences and/or light and heavy chain variable sequences as any of the antibodies in Antibody Set I or Antibody Set II.
• detection antibodies are conjugated to a detection particle, including but not limited to a gold nanoparticle, latex bead, fluorescent label, enzyme, colloidal metal, or magnetic particle.
• the gold nanoparticle may be a nanoshell or a nanosphere.
• the detection particle can have any suitable size.
• a gold nanoparticle may be about 20 nm, 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 250 nm, or 300 nm in diameter.
• the detection antibodies are comprised within a conjugate release area.
• the detection antibodies are pre-loaded onto the device at the time of manufacture and/or packaging.
• an end user e.g., a doctor or clinician, applies the detection antibodies to the device prior to running an LFA.
• the detection antibodies may be provided with the device, for example in a kit as set forth below.
• the devices provided herein comprise one or more capture antibodies provided herein, including any of the antibodies in Antibody Set I or Antibody Set II, or capture antibodies that bind the same antigen and/or share the same CDR sequences and/or light and heavy chain variable sequences as any of the antibodies in Antibody Set I or Antibody Set II.
• the capture antibodies are comprised at a test line of the device.
• the devices provided herein are pre-loaded with capture antibodies at the test line.
• an end user e.g., a doctor or clinician, applies the capture antibodies to the test line prior to running an LFA.
• the capture antibodies may be provided with the device, for example in a kit as set forth below.
• the devices provided herein comprise a control line comprising one or more control antibodies that bind to the conjugated detection antibodies.
• the control antibodies can include anti-human immunoglobulin G (IgG) antibodies produced in goat (goat anti-human IgGs).
• IgG anti-human immunoglobulin G
• the control line confirms that a test performed on the LFA is working properly.
• the devices provided herein comprise the control line comprising gold nanoparticles conjugated to streptavidin (Au-streptavidin).
• Au-streptavidin conjugated to streptavidin
• a mixture of Au-streptavidin and a blue dye can be dispensed at the control line.
• the gold nanoparticles can have a diameter of 40 nanometers, and the concentration of Au-streptavidin can be 20 OD.
• Au-streptavidin with the blue dye can be dispensed at a rate of 0.08 microliters per millimeters.
• the blue dye can be diluted to a 1 : 100 ratio.
• the gold nanoparticles can attach at the control line via interactions of streptavidin to a nitrocellulose membrane surface of the device. When a sample is loaded and ran on the device, the blue dye washes off and a red color from the gold nanoparticles at the control line appears, which confirms that a test performed on the LFA
• kits comprising one or more of the antibodies or antigen-binding fragments disclosed herein.
• the kits comprise isolated antibodies for application to a device for use in LFAs.
• the kits comprise a device provided herein, e.g., a strip, cartridge, or cassette, wherein the antibodies are already affixed.
• kits can be configured for use in a variety of settings for preparing or running a SARS-CoV-2 detection assay.
• the kits are configured for home use.
• the kits are configured for use in a laboratory.
• the kits are configured for use in a clinical setting (e.g., a hospital).
• kits provided herein may comprise additional reagents for use in preparing or running a SARS-CoV-2 detection assay.
• the kit may include a lysis buffer, a running buffer, control antibodies (e.g., goat anti-human IgG), nanoparticles for antibody conjugation, a positive sample, a negative sample, and any other suitable reagent for use in LFAs.
• the lysis buffer can include phosphate buffer saline (PBS) and Tween 20 mixture (e.g., IX PBS and 1% Tween 20), RIPA (e.g., 50 nM Tris, 150 mM sodium chloride, 1% Triton X100, 0.5% sodium deoxycholate, and 0.1% SDS), viral RNA buffer (e.g., Zymo Research RNA buffer and 0.5% DDT), Fantibody, Tris and Triton X100 mixture (e.g., 50 mM Tris, 150 mM sodium chloride, and 1% Triton X100), and any other suitable lysis buffer for use in LFAs.
• PBS phosphate buffer saline
• Tween 20 mixture e.g., IX PBS and 1% Tween 20
• RIPA e.g., 50 nM Tris, 150 mM sodium chloride, 1% Triton X100, 0.5% sodium deoxycholate, and 0.1% SDS
• the running buffer can include 300 mM Tris, 1.5x PBS, 1.5% PVP-40, 30 mM EDTA, 1% Tween 20, 0.5% Digested Casein, and 0.05% Sodium Azide.
• the running buffer can include a phosphate-NaCl buffer solution (PBS) and/or Tween 20.
• PBS phosphate-NaCl buffer solution
• the running buffer may have a pH of approximately 6.4, 7.4, 8.4, or any suitable pH for use in LFAs.
• the pH of the running buffer is approximately 7.4.
• the positive sample can include attenuated vims, such as a heat inactivated vims (e.g., a SARS-CoV-2 Spike protein recombinant antigen heat inactivated SARS-CoV-2 virus).
• the negative sample can include the mnning buffer (e.g., no attenuated vims).
• kits Provided herein in certain embodiments are methods of diagnosing SARS-CoV-2 in a subject using the antibodies, compositions, devices, and/or kits provided herein.
• the methods provided herein comprise obtaining a biological sample, applying the biological sample to an LFA device as provided herein, and detecting the presence of SARS-CoV-2 N protein in order to diagnose SARS-CoV-2 infection.
• the biological sample is loaded onto the sample application area of an LFA strip, cartridge, or cassette, and an LFA is performed.
• Running buffer is applied to the strip, cartridge, or cassette, and the sample flows across the conjugate release area, which comprises one or more detection antibodies provided herein conjugated to a detection particle, and then across the reaction area, which comprises a test line comprising one or more capture antibodies provided herein and a control line comprising one or more control antibodies or an Au-streptavidin with blue dye mixture.
• the presence or absence of a signal at the test and control line is used to diagnose SARS-CoV-2 infection based on the presence of SARS-CoV-2 N protein. For example, a signal at both the control line and the test line indicates a positive test, while a signal at the control line without a signal at the test line indicates a negative test.
• the methods provided herein provide immediate or near immediate, e.g., within seconds, minutes, or less than an hour diagnosis.
• the methods provided herein may be performed in a variety of settings, including a medical setting such as a hospital, clinic, or laboratory or in a home setting.
• a biological sample to be analyzed using the devices, kits, and methods provided herein may be a saliva sample, a sample from the anterior nares, mid turbinate, nasopharynx or the oropharynx, whole blood, blood serum, plasma, semen, feces, sputum, mucus, urine, amniotic fluid, pleural effusions and ascites, cerebrospinal fluid, or a tissue sample.
• Example 1 Global screening of antibody combination pairs for SARS-CoV-2 nucleocapsid protein recombinant antigen binding using ELISA
• Antibody combinations were screened using an enzyme-linked immunosorbent assay (ELISA) for binding to a SARS-CoV-2 nucleocapsid protein recombinant antigen (recombinant N protein).
• ELISA enzyme-linked immunosorbent assay
• the ELISA procedure included the following steps. 100 pL of a capture antibody was added to each well of a microplate and incubated for approximately 48 hours at 4 °C. Each well was washed three times using 300 pL of a wash buffer each time. 250 pL of Thermo Super Block was added to each well of the microplate and incubated while shaking for approximately 1 to 2 hours at 4 °C. Each well was washed three times using 300 pL of a wash buffer each time. In a positive sample, 100 pL of a recombinant N protein with a polyhistidine- tag derived from human embryonic kidney (HEK) cells provided by GeneTex was added to each well of the microplate.
• HEK human embryonic kidney
• a global antibody screening was first performed to test 484 antibody combinations. As shown in Table 2, a pairing matrix of 22 capture antibodies and 22 detection antibodies, totaling 484 combinations (or pairs), was screened against the recombinant N protein at a concentration of 10 ng/mL. Eight detection antibodies and ten capture antibodies were identified with high signal-to-noise ratios.
• the eight detection antibodies included Sinobiological 40143- MM08 (“MM08” or “MM8”), Exonbio 3E6 (“3E6”), Exonbio 3H1 (“3H1”), Exonbio 3H6 (“3H6”), Exonbio 5B1 (“5B1”), Exonbio 5C3 (“5C3”), Exonbio 5E2 (“5E2”), and Exonbio 6F2 (“6F2”).
• the ten capture antibodies included Sinobiological 40143-MM05 (“MM05” or “MM5”), MM08, GeneTex 5686 (“5686”), GeneTex 5712 (“5712”), GeneTex 5689 (“5689”), Exonbio IAS (“IAS”), Exonbio 3E6 (“3E6”), Exonbio 3H1 (“3H1”), Exonbio 5B1 (“5B1”), and Exonbio 5C3 (“5C3”) (selected detection and capture antibodies highlighted in Table 2).
• Table 2 Signal-to-noise ratios of 22 by 22 antibody pairing matrix.
• Example 2 Targeted screening of antibody combination pairs for recombinant N protein binding using ELISA
• a targeted screening of the top detection and capture antibodies identified in the global screening was performed. As shown in Table 3, a pairing matrix of 10 capture antibodies by 8 detection antibodies, totaling 80 combinations, was screened against the recombinant N protein at a concentration (1 ng/mL) lower than in the global antibody screening.
• the 14 antibody pairs identified in the targeted screening were further characterized by determining their limit of detection (LoD) and ranking each antibody based on each pair’s ability to detect attenuated vims.
• LoD limit of detection
• Table 4 the viral titer detectable by each of the 14 antibody pairs was quantified based on fifty-percent tissue culture infective dose (TCID50) using heat inactivated vims (e.g., attenuated vims) at 4000- and 16000-fold dilutions.
• the four identified top antibody pairs were selected for feasibility testing with a lateral flow assay (LFA).
• LFA lateral flow assay
• the six selected antibody pairs with two antibody orientations were tested with heat inactivated vims at 100-fold concentrations (3.8 x 10 4 TCID50/mL), as shown in Figure 2, in concentrations ranging from 5 ng/mL to 20 ng/mL.
• no heat inactivated virus was added and a buffer (IX PBS plus 1% TW20) was added instead.
• the capture antibody was striped on nitrocellulose at a test line (TL) and the detection antibody was coupled to 150 nm gold nanoparticles.
• Goat anti-human IgGs was immobilized at the control line (CL).
• One of R04, MM05, MM08, 5B1, or 3H1 was immobilized at the TL.
• the antibody pairs that resulted in signal at the TL in the positive sample with the heat inactivated virus and no or low signal at the TL in the negative sample were identified (i.e., high signal-to-noise ratios).
• the antibody pairs identified with high signal-to-noise ratios included MM05-R04, MM05-5B1, MM08-R04, MM08-5B1, and 5B1-MM08 (detection antibody-capture antibody).
• Table 5 Standard curve data on LFA of twelve selected antibody pairs
• Example 5 Optimizing pH flex to increase LFA sensitivity for screening antibody binding
• Optimal pH conditions for increased assay sensitivity was determined by performing a pH flex procedure. Conjugation optimization was performed with the following antibodies: R04, MM05, and 5B1. A reaction buffer pH sweep was performed using phosphate-buffered saline (PBS) at pH 6.4, 7.4 and 8.4. Concentration of the antibody MM08 immobilized at the TL was increased to 2 mg/mL compared to the concentration of 1 mg/mL used in Example 4. Goat anti-human IgGs was immobilized at the CL. In the positive sample, heat inactivated virus at 100-fold dilution was added. In the negative sample, no heat inactivated virus was added. As shown in Figure 6, 5B1 and R04 at pH 7.4 showed largest signal-to-noise ratios at the TL. Thus, pH 7.4 was determined to be the optimal condition for LFA.
• PBS phosphate-buffered saline
• the ExonBio 3E6 antibody was tested with the recombinant N protein using LFA to identify target antibody pairs.
• antibody pairs were tested with heat inactivated virus at a concentration of 10 ng/mL.
• no heat inactivated virus was added.
• Goat anti-human IgGs was immobilized at CL.
• One of R04, MM08, 5B1, 5C3, or 3E6 was immobilized at the TL.
• the MM08-R04 antibody pair was used as a control antibody pair. As shown in Figure 8, MM08-3E6, 5B1-3E6, and 5B1-R04 yielded high signal- to-noise ratios.
• Example 7 The top three antibody pairs (MM08-3E6, 5B1-3E6, and 5B1-R04) identified in Example 7 and the control antibody pair (MM08-R04) was tested to perform recombinant N protein titration.
• Recombinant N protein at concentrations of 0.1, 0.5, 1, and 5 ng/mL of was used to perform the titration.
• MM08-3E6 and 5B1-3E6 showed high sensitivity to recombinant N protein binding, whereas MM08-R04 and 5B1-R04 showed low sensitivity.
• Antibody pairs MM08-3E6 and 5B1-3E6 binding was further characterized using a new lot of 3E6 conjugated gold nanoshells (QXD321) compared to an old lot of 3E6 conjugated gold nanoshells (QXD329).
• QXD321 3E6 conjugated gold nanoshells
• recombinant N protein was immobilized at the TL at a concentration of 1 ng/mL.
• negative sample no recombinant N protein was immobilized.
• 5B1-3E6 showed non-specific binding, such as binding at the TL in both the positive and negative sample. Binding of MM08-3E6 using the old lot and new lot was comparable.
• Example 10 Evaluating lysis buffer options with MM08-3E6 and recombinant antigen
• Lysis buffer options were evaluated for MM08-3E6 binding with recombinant N protein.
• the lysis buffer options tested includes: PBS and Tween 20 mixture (IX PBS and 1% Tween 20) as the control, RIPA (50 nM Tris, 150 mM sodium chloride, 1% Triton X100, 0.5% sodium deoxycholate, and 0.1% SDS), viral RNA buffer (buffer and 0.5% DDT), Fantibody, and Tris and Triton X100 mixture (50 mM Tris, 150 mM sodium chloride, and 1% Triton X100).
• the procedure for performing the evaluation includes creating a mixture of 20 pL of the recombinant N protein at a concentration of 1 ng/mL with 35 pL of the lysis buffer, adding 10 pL of a 3E6-bound conjugate to the LFA test strip with MM08 bound at the TL, and running the recombinant N protein and lysis buffer mixture on an LFA test strip without chasing with a running buffer.
• minimal interference was seen with Fantibody and the Tris and Triton X100 mixture, whereas commercial RNA extraction buffer (Zymo Research) and RIPA affected the sample negatively.
• Example 11 Optimization of an amount of conjugated particles and sample volume flex for MM08-3E6
• Example 12 Clinical testing of antibody pairs with human saliva samples
• MM08-3E6 and MM08-R4 antibody pairs were evaluated for detecting recombinant N protein with human saliva samples.
• Human saliva samples were provided by Lee BioSolutions.
• Ten human saliva samples (e.g., clinical samples) with a varying range of viral loads (cycle time, Ct), which is related to the number of days from symptom onset of the human patient (*Days), were used in the evaluation.
• cycle time, Ct cycle time
• Example 13 Qualification of various COVID-19 strains for MM08-3E6 binding
• Binding of MM08-3E6 was tested against various strains of COVID-19.
• the COVID-19 included: 1) the SARS-related Coronavirus-2, culture fluid, heat inactivated, isolate: USA-WA1/2020, category number 0810587CFHI (“Washington strain”), 2) SARS-related Coronavirus-2, culture fluid, heat inactivated, isolate: Italy-INMIl, category number 0810589CFHI (“Italy strain”), and 3) SARS-related Coronavirus-2, culture fluid, heat inactivated, isolate: Hong Kong/VM20001061/2020, category number 0810590CFHI (“Hong Kong strain”).
• the three strains were diluted to TCID50/mL values including 3.84 x 10 4 , 7.9 x 10 3 , and 3.8 x 10 3 and run on LFA test strips immobilized with MM08-3E6.
• Zeptometrix determined the stock TCID50/mL concentration using the same cell line (VERO E6), a kidney cell line. As shown in Figures 16 and 17 and Table 6, the Washington strain, Italy strain, and Hong Kong strain were detected using LFA test strips loaded with MM08-3E6.
• Example 14 Determining the limit of detection (LoD) of recombinant antigen
• Example 16 Clinical testing of antibody pairs with human nasal swab samples
• Antibody combinations were evaluated for detection of recombinant N protein with human nasal swab samples.
• Human nasal swab samples were provided by Reprocell and Lee BioSolutions.
• Four Reprocell nasal swab samples 13, 14, 18, and 20
• four Lee BioSolutions nasal swab samples 86, 53, 55, 54
• Ct viral load
• Lee BioSolutions samples varied in viral load (Ct).
• LFA cassettes were evaluated for sensitivity of antibody binding with human nasal swab samples.
• Antibody combinations that were evaluated on the LFA cassettes included 3E6/(MM08+48), (3E6+47)/(MM08+48), 3E6/MM08, and (3E6+47)/48.
• 3E6/(MM08+48) 3E6 was loaded at the TL, and both MM08 and 48 were loaded as conjugates.
• both 3E6 and 47 were loaded at the TL, and both MM08 and 48 were loaded as conjugates.
• For the antibody combination 3E6/MM08 3E6 was loaded at the TL, and MM08 was loaded as a conjugate.

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