DEVICES, METHODS, AND KITS FOR DIAGNOSING SARS COV-2 INFECTION

CROSS-REFERENCE TO RELATED APPLICATION'S )

[0001] The present application claims priority to U.S. Provisional Application No. 63/176,830, filed April 19, 2021, and titled “DEVICES, METHODS, AND KITS FOR DIAGNOSING SARS COV-2 INFECTION,” the disclosure of which is incorporated by reference herein in its entirety.

TECHNICAL FIELD

[0002] The present technology is related to devices, methods, and kits for diagnosing a subject with SARS-CoV-2 vims (“COVID-19”) infection.

BACKGROUND

[0003] The worldwide pandemic caused by the spread of COVID-19 has killed over 1,400,000 persons worldwide as of November 2020. It has also impaired the quality of life of many others and had significant social and economic impacts across the world. Diagnostic testing for infection is central to detecting the virus in persons presenting with and without COVID-19 symptoms and those who have been in contact with persons exposed to COVID-19 to contain the spread of the virus. Diagnostic testing will continue to be important after a vaccine is approved and distributed as it will be important to track the effectiveness of such vaccines and it is expected to take several months to widely distribute the vaccine. Furthermore, a large number of people do not want to get vaccinated, and vaccines are not 100% effective. It will continue to be important to quarantine people who are infected.

[0004] Current diagnostic testing requires individuals to go to testing sites and provide a sample, which is usually a specimen from the nasopharynx or oropharynx. For example, the reverse transcriptase polymerase chain reaction (RT-PCR) test is the predominate test used to detect COVID-19 with a high degree of accuracy. 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. SUMMARY

[0005] 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, shares the same CDR sequences, and/or shares the same light and heavy chain variable sequences as Exonbio 3E6; and Meridian 9547 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 9547; c) 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 detection antibody; 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 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; d) Sinobiological 40143-MM08 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 Sinobiological 40143-MM08; 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 a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 3E6; and Meridian 9547 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 9547 ; e) 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; 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 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; f) Sinobiological 40143-MM08 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 Sinobiological 40143-MM08; 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; and Exonbio 3E6 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 Exonbio 3E6; g) 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 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; h) Sinobiological 40143-MM08 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 Sinobiological 40143-MM08; and Exonbio 3E6 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 Exonbio 3E6; i) Exonbio 5B 1 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 5B1; and Exonbio 3E6 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 Exonbio 3E6; j) 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 Exonbio 5B1 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 Exonbio 5B1; k) Exonbio 5C3 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 5C3; and Exonbio 3E6 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 Exonbio 3E6; l) Exonbio 3E6 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 3E6; and Exonbio 5C3 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 Exonbio 5C3; m) Exonbio 5B 1 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 5B1; and Sinobiological 40143-R004 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-R004; n) Sinobiological 40143-R004 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Sinobiological 40143-R004; and Exonbio 5B1 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 Exonbio 5B1; o) Exonbio 5B 1 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 5B1; and Sinobiological 40143-MM05 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-MM05; p) Sinobiological 40143-MM05 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Sinobiological 40143-MM05; and Exonbio 5B1 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 Exonbio 5B 1 ; q) Exonbio 5B 1 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 5B1; and 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; r) Sinobiological 40143-MM08 detection antibody or a detection 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 Exonbio 5B1 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 Exonbio 5B 1 ; s) GeneTex 5712 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as GeneTex 5712; and Exonbio 3E6 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 Exonbio 3E6; t) Exonbio 3E6 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 3E6; and GeneTex 5712 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 GeneTex 5712; u) GeneTex 5712 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as GeneTex 5712; and Exonbio 3H1 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 Exonbio 3H1; v) Exonbio 3H1 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 3H1; and GeneTex 5712 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 GeneTex 5712; w) Exonbio 3H1 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 3H1; and Sinobiological 40143-MM05 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-MM05; x) Sinobiological 40143-MM05 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Sinobiological 40143-MM05; and Exonbio 3H1 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 Exonbio 3H1; y) 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 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; z) 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; and Exonbio 3E6 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 Exonbio 3E6; aa) 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 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 bb) Sinobiological 40143-MM08 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 Sinobiological 40143-MM08; Exonbio 3E6 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 Exonbio 3E6; and Meridian 9547 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 9547.

[0006] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the device is a strip, cassette, or cartridge.

[0007] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the device comprises a sample application area, a conjugate release area, and a reaction area.

[0008] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, 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; e) Exonbio 5C3 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 5C3; f) Sinobiological 40143-MM05 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-MM05; g) GeneTex 5712 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 GeneTex 5712; h) Meridian 9547 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; i) Meridian 9548 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; and j) Sinobiological 40143-R004 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-R004; wherein the one or more detection antibodies are conjugated to a detection particle. [0009] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the detection particle is selected from the group consisting of gold nanoparticle, latex bead, fluorescent label, enzyme, colloidal metal, and magnetic particle.

[0010] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, 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 ; e) Exonbio 5C3 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 5C3; f) Sinobiological 40143-MM05 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-MM05; g) GeneTex 5712 or a capture antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as GeneTex 5712; h) Meridian 9547 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 9547; i) Meridian 9548 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; and j) Sinobiological 40143-R004 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 Sinobiological 40143-R004.

[0011] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the reaction area comprises a control line comprising one or more control antibodies that bind the detection antibody.

[0012] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the control antibody is an anti-human immunoglobulin G (IgG) antibody.

[0013] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, a concentration of the particles conjugated with the capture antibody set is in the range of approximately 20 to 30 optical density units.

[0014] In various embodiments, 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.

[0015] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the biological sample is saliva.

[0016] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the biological sample is from a nasal cavity of the subject.

[0017] In various embodiments, 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 heavy chain variable sequences as Exonbio 3E6; and Meridian 9547 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 9547; c) 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 detection antibody; 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 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; d) Sinobiological 40143-MM08 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 Sinobiological 40143-MM08; 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 a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 3E6; and Meridian 9547 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 9547 ; e) 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; 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 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; f) Sinobiological 40143-MM08 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 Sinobiological 40143-MM08; 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; and Exonbio 3E6 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 Exonbio 3E6; g) 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 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; h) Sinobiological 40143-MM08 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 Sinobiological 40143-MM08; and Exonbio 3E6 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 Exonbio 3E6; i) Exonbio 5B 1 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 5B1; and Exonbio 3E6 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 Exonbio 3E6; j) 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 Exonbio 5B1 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 Exonbio 5B1; k) Exonbio 5C3 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 5C3; and Exonbio 3E6 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 Exonbio 3E6; l) Exonbio 3E6 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 3E6; and Exonbio 5C3 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 Exonbio 5C3; m) Exonbio 5B 1 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 5B1; and Sinobiological 40143-R004 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-R004; n) Sinobiological 40143-R004 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Sinobiological 40143-R004; and Exonbio 5B1 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 Exonbio 5B1; o) Exonbio 5B 1 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 5B1; and Sinobiological 40143-MM05 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-MM05; p) Sinobiological 40143-MM05 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Sinobiological 40143-MM05; and Exonbio 5B1 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 Exonbio 5B 1 ; q) Exonbio 5B 1 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 5B1; and 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; r) Sinobiological 40143-MM08 detection antibody or a detection 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 Exonbio 5B1 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 Exonbio 5B 1 ; s) GeneTex 5712 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as GeneTex 5712; and Exonbio 3E6 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 Exonbio 3E6; t) Exonbio 3E6 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 3E6; and GeneTex 5712 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 GeneTex 5712; u) GeneTex 5712 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as GeneTex 5712; and Exonbio 3H1 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 Exonbio 3H1; v) Exonbio 3H1 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 3H1; and GeneTex 5712 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 GeneTex 5712; w) Exonbio 3H1 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Exonbio 3H1; and Sinobiological 40143-MM05 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-MM05; x) Sinobiological 40143-MM05 detection antibody or a detection antibody that binds a same antigen, shares same CDR sequences, and/or shares same light and heavy chain variable sequences as Sinobiological 40143-MM05; and Exonbio 3H1 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 Exonbio 3H1; y) 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 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; z) 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; and Exonbio 3E6 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 Exonbio 3E6; aa) 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 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 bb) Sinobiological 40143-MM08 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 Sinobiological 40143-MM08; Exonbio 3E6 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 Exonbio 3E6; and Meridian 9547 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 9547.

[0018] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the kit further comprises a lateral flow immunochromatographic assay (LFA) device.

[0019] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the one or more combinations of antibodies are packaged separately from the LFA device.

[0020] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the one or more combinations of antibodies are pre-loaded onto the device.

[0021] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the device comprises a sample application area, a conjugate release area, and a reaction area.

[0022] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the reaction area comprises a test line.

[0023] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, capture antibodies from the one or more combinations of antibodies are pre-loaded onto the test line.

[0024] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, detection antibodies from the one or more combinations of antibodies are pre-loaded onto the conjugate release area.

[0025] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the kit further comprises a detection particle. [0026] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the detection particle is packaged separately from the one or more combinations of antibodies.

[0027] In one embodiment, which may be used or combined with any of the above or below mentioned embodiments, the detection particle is pre-conjugated to a detection antibody from the one or more combinations of antibodies.

[0028] In various embodiments, 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.

[0029] In various embodiments, 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.

[0030] In various embodiments, 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:

[0031] These and other embodiments of the present invention will be disclosed in further detail herein below.

BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 illustrates standard curves of antibody pairs evaluated with an enzyme- linked immunosorbent assay (ELISA).

[0033] Figure 2 illustrates antibody pairs in two antibody orientations selected for feasibility testing with a lateral flow assay (LFA).

[0034] Figure 3 illustrates an image of an LFA test strip loaded with the antibody pairs of

Figure 2. [0035] Figure 4 illustrates standard curves of the antibody pairs of Figure 2 evaluated with the LFA.

[0036] Figure 5 illustrates standard curves of a subset of the antibody pairs of Figure 2 evaluated with the LFA.

[0037] Figure 6 illustrates an image of an LFA test strip loaded with antibody pairs under varying pH conditions.

[0038] Figure 7 illustrates images of LFA test strips loaded with antibody pairs under varying EDC/Sulfo-NHS ratios.

[0039] Figure 8 illustrates an image of an LFA test strip loaded with antibody pairs.

[0040] 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).

[0041] Figure 10 is a graph of signal intensities at the test line (TL) of the LFA test strips of Figure 9.

[0042] Figure 11 illustrates an image of an LFA test strip loaded with antibody pairs.

[0043] Figure 12 illustrates an image of an LFA test strip loaded with an antibody pair and tested with various lysis buffers.

[0044] 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.

[0045] 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.

[0046] Figure 15 illustrates images of LFA test strips loaded with antibody pairs for evaluating detection of recombinant N protein from human saliva samples.

[0047] Figure 16 illustrates images of LFA test strips loaded with an antibody pair for evaluating detection of various strains of COVID-19.

[0048] Figure 17 is a graph of signal intensities at the TL of the LFA test strips of Figure 16. [0049] 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).

[0050] 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.

[0051] Figure 20 illustrates images of LFA test strips loaded with antibody pairs for evaluating detection of recombinant N protein from human nasal swab samples.

[0052] Figure 21 illustrates images of LFA cassettes loaded with antibody pairs for evaluating detection of recombinant N protein from human nasal swab samples.

DETAILED DESCRIPTION

Definitions

[0053] The term “antigen” as used herein 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).

[0054] The term “antibody” as used herein 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. In certain embodiments, an antibody may be monoclonal or polyclonal, and may fall into any immunoglobulin class (e.g., IgG, IgA, IgM) or subclass.

Overview

[0055] The present technology is directed to compositions, methods, and kits for diagnosing a subject with SARS-CoV-2 vims.

[0056] As disclosed herein, various antibody combinations were screened for sensitivity for detecting a SARS-CoV-2 nucleocapsid protein recombinant antigen (recombinant N protein). For the initial global screening, combinations of 22 capture antibodies and 22 detection antibodies were screened using an enzyme-linked immunosorbent assay (ELISA). In a subsequent targeted screening, select antibody pairs from the global screen that detected the recombinant N protein with a high-signal-to-noise ratio, including 10 capture antibodies and 8 detection antibodies, were further screened using ELISA and a concentration of the recombinant N protein lower than that used in the global screening. Select antibody pairs from the targeted screening that detected the recombinant N protein with a high-signal-to-noise ratio, including 14 antibody pairs, were further characterized by determining their limit of detection (LoD) and ranking each antibody pair based on ability to detect recombinant N protein and attenuated virus (e.g., heat inactivated SARS-CoV-2 vims).

[0057] 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.

[0058] The process outlined above resulted in the identification of select antibody pairs with high sensitivity for isolated SARS-CoV-2 N protein and the ability to detect isolated recombinant N protein as well as the ability to detect N protein in attenuated virus and clinical samples. These antibody pairs provide accurate detection of SARS-CoV-2 with a high signal- to-noise ratio.

[0059] Accordingly, provided herein are 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.

[0060] In certain embodiments, the compositions, devices, and kits provided herein comprise one or antibodies selected from:

• Sinobiological 40143-MM08 (“MM08” or “MM8”; https://www.sinobiological.com/antibodies/cov-nucleocapsid-4 0143-mm08),

• Exonbio 3E6 (“3E6”),

Exonbio 3H1 (“3H1”), • Exonbio 5B1 (“5B1”),

• Exonbio 5C3 (“5C3”),

• Sinobiological 40143-MM05 (“MM05” or “MM5”; https://www.sinobiological.com/antibodies/cov-nucleocapsid-4 0143-mm05),

• GeneTex 5712 (“5712”; https://www.genetex.com/Product/Detail/SARS-CoV-2- COVID-19-nucleocapsid-antibody-HL455-MS/GTX635712),

• Sinobiological 40143-R004 (“R004”; https://www.sinobiological.com/antibodies/cov- nucleocapsid-40143 -r004) ,

• Meridian 9547 (“9547” or “47”; https://meridianlifescience.com/sars-cov2- nucleocapsid-pairs), and

• Meridian 9548 (“9548” or “48”; https://meridianlifescience.com/sars-cov2- nucleocapsid-pairs),

• or antibodies that specifically bind the same antigen or epitope as any of the aforementioned antibodies or share the CDR sequences or variable heavy and light chain sequences of the aforementioned antibodies.

In certain embodiments, the one or more antibodies may be incorporated into a fusion protein.

[0061] In certain embodiments of the compositions, devices, and kits provided herein, the antibodies are paired as set forth in Table 1. In certain embodiments, the antibodies in Set I are capture antibodies and the antibodies in Set II are detection antibodies. In other embodiments, the antibodies in Set I are detection antibodies and the antibodies in Set II are capture antibodies.

Table 1

Compositions

[0062] Provided herein in certain embodiments are compositions comprising two or more of the antibodies disclosed herein. In certain of these embodiments, the compositions comprise a capture antibody set and a detection antibody set.

[0063] 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.

[0064] In certain embodiments, the compositions provided herein comprise Meridian 9548, Exonbio 3E6, and Meridian 9547. For example, the composition may include Meridian 9548 as the capture antibody and Exonbio 3E6 and Meridian 9547 as the detection antibodies. In another example, the composition may include Meridian 9548 as the detection antibody and Exonbio 3E6 and Meridian 9547 as the capture antibodies. In certain embodiments, 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. [0065] In certain embodiments, the compositions provided herein comprise Sinobiological 40143-MM08, Meridian 9548, Exonbio 3E6, and Meridian 9547. For example, the composition may include Sinobiological 40143-MM08 and Meridian 9548 as the capture antibodies and Exonbio 3E6 and Meridian 9547 as the detection antibodies. In another example, the composition may include Sinobiological 40143-MM08 and Meridian 9548 as the detection antibodies and Exonbio 3E6 and Meridian 9547 as the capture antibodies. In certain embodiments, 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.

[0066] In certain embodiments, the compositions provided herein comprise Exonbio 3E6, Sinobiological 40143-MM08, and Meridian 9548. For example, the composition may include Exonbio 3E6 as the capture antibody and Sinobiological 40143-MM08 and Meridian 9548 as the detection antibodies. In another example, the composition may include Exonbio 3E6 as the detection antibody and Sinobiological 40143-MM08 and Meridian 9548 as the capture antibodies. In certain embodiments, 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.

[0067] In certain embodiments, the compositions provided herein comprise Exonbio 3E6 and Sinobiological 40143-MM08. For example, the composition may include Exonbio 3E6 as the capture antibody and Sinobiological 40143-MM08 as the detection antibody. In another example, the composition may include Exonbio 3E6 as the detection antibody and Sinobiological 40143-MM08 as the capture antibody. In certain embodiments, 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.

[0068] In certain embodiments, the compositions provided herein comprise Sinobiological 40143-MM08, Exonbio 3E6, and Meridian 9547. For example, the composition may include Sinobiological 40143-MM08 as the capture antibody and Exonbio 3E6 and Meridian 9547 as the detection antibodies. In another example, the composition may include Sinobiological 40143-MM08 as the detection antibody and Exonbio 3E6 and Meridian 9547 as the capture antibodies. In certain embodiments, 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

[0069] Provided herein in certain embodiments are devices for use in LFAs comprising two or more of antibodies provided herein. In certain embodiments, these devices comprise a composition provided herein. For example, in certain embodiments, 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.

[0070] In certain embodiments, the devices provided herein comprise strips, cartridges, cassettes, or any other suitable substrate for use in an LFA. In certain of these embodiments, 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. In certain of these embodiments, the device is a dual line device comprising a test line and a control line in the reaction area.

[0071] In certain embodiments, the sample application area may comprise a sample pad comprising, but not limited to, woven mesh or cellulose fiber. In certain embodiments the conjugate release area may comprise a conjugate pad comprising, but not limited to, non-woven glass fiber.

[0072] In certain embodiments, 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. These 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. For example, 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. In certain embodiments, the detection antibodies are comprised within a conjugate release area. In certain embodiments, the detection antibodies are pre-loaded onto the device at the time of manufacture and/or packaging. In other embodiments, an end user, e.g., a doctor or clinician, applies the detection antibodies to the device prior to running an LFA. In these embodiments, the detection antibodies may be provided with the device, for example in a kit as set forth below.

[0073] In certain embodiments, 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. In certain embodiments, the capture antibodies are comprised at a test line of the device. In certain of these embodiments, the devices provided herein are pre-loaded with capture antibodies at the test line. In other embodiments, an end user, e.g., a doctor or clinician, applies the capture antibodies to the test line prior to running an LFA. In these embodiments, the capture antibodies may be provided with the device, for example in a kit as set forth below.

[0074] In certain embodiments, the devices provided herein comprise a control line comprising one or more control antibodies that bind to the conjugated detection antibodies. For example, the control antibodies can include anti-human immunoglobulin G (IgG) antibodies produced in goat (goat anti-human IgGs). The control line confirms that a test performed on the LFA is working properly.

[0075] In certain embodiments, the devices provided herein comprise the control line comprising gold nanoparticles conjugated to streptavidin (Au-streptavidin). For example, 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 is working properly.

Kits

[0076] Provided herein in certain embodiments are kits comprising one or more of the antibodies or antigen-binding fragments disclosed herein. In certain embodiments, the kits comprise isolated antibodies for application to a device for use in LFAs. In other embodiments, the kits comprise a device provided herein, e.g., a strip, cartridge, or cassette, wherein the antibodies are already affixed.

[0077] The kits can be configured for use in a variety of settings for preparing or running a SARS-CoV-2 detection assay. In certain embodiments, the kits are configured for home use. In other embodiments, the kits are configured for use in a laboratory. In certain embodiments, the kits are configured for use in a clinical setting (e.g., a hospital).

[0078] In certain embodiments, the kits provided herein may comprise additional reagents for use in preparing or running a SARS-CoV-2 detection assay. For example, 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. 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. In some embodiments, the running buffer can include a phosphate-NaCl buffer solution (PBS) and/or Tween 20. The running buffer may have a pH of approximately 6.4, 7.4, 8.4, or any suitable pH for use in LFAs. In some embodiments, 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).

Methods

[0079] 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.

[0080] In certain embodiments, 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. In certain of these embodiments, 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.

[0081] In certain embodiments, the methods provided herein provide immediate or near immediate, e.g., within seconds, minutes, or less than an hour diagnosis.

[0082] 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.

[0083] In certain embodiment, 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.

EXAMPLES

Example 1: Global screening of antibody combination pairs for SARS-CoV-2 nucleocapsid protein recombinant antigen binding using ELISA

[0084] 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).

[0085] Generally, 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. In a negative sample, 100 pL of assay buffer (no recombinant N protein) was added to each well of the microplate. The sample was incubated while shaking for approximately 1 hour at 4 °C. Each well was washed three times using 300 pL of a wash buffer each time. 100 pL of avidin horseradish peroxidase (Av-HRP) was added to each well of the microplate and incubated while shaking for 30 minutes at 4 °C. Each well was washed five times using 500 pL of a wash buffer each time. 100 pL of Step Ultra TMB Substrate was added to each well and incubated at room temperature (approximately 20 °C) for approximately 10 to 15 minutes while avoiding light exposure. 50 pL of 2 molar sulfuric acid was added to terminate the reaction. Absorbance of the sample in each well was measured at 450 nm to 570 nm for detection of antigen binding. Signal-to-noise ratios were calculated as the ratio of signal from the positive sample to the signal from the negative sample.

[0086] 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

[0087] 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. 14 antibody pairs were identified based on high signal-to-noise ratios: 3E6-5712, 3H1-5712, 3H6-5712, 5B1- 1A5, 3H1-3E6, 5B1-3E6, 5C3-3E6, 6F2-3E6, MM06-3E6, 3H1-MM05, 3H6-MM05, 5B1- MM05, MM08-MM05, and 5B1-MM08 (detection antibody-capture antibody) (selected pairs highlighted in Table 3).

Table 3: Signal-to-noise ratios of 10 by 8 antibody pairing matrix Example 3: Characterization of antibody combination pairs for recombinant N protein binding using ELISA

[0088] 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. As shown in 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.

Table 4: Viral titers, signal-to-noise ratios, signal-minus-noise ratios, and LoD for 14 antibody pairs

[0089] As shown in Figure 1, standard curves were determined with the recombinant N protein at a concentration ranging from 0.02 ng/mL to 1 ng/mL. Signal-to-noise ratios and signal-minus-noise values were calculated for each of the 14 antibody pairs. Top antibody pairs were identified based on material availability and performance (e.g., having high signal-to-noise ratio). The four identified top antibody pairs included MM08-MM05, 5B1-MM05, 3H1-MM05, and 5B1-MM08 (detection antibody-capture antibody) (highlighted in Table 4). Example 4: Testing lateral flow assay (LFA) feasibility of antibody pairs identified from ELISA- based screening and characterizations

[0090] The four identified top antibody pairs, along with two additional pairs (SinoBiological 40143-R004 (“R04” or “R4”)-MM08 and R04-MM05), were selected for feasibility testing with a lateral flow assay (LFA). In the positive sample, 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. In the negative sample, no heat inactivated virus was added and a buffer (IX PBS plus 1% TW20) was added instead.

[0091] For each antibody pair, 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. As shown in Figure 3, 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).

[0092] As shown in Table 5 and Figures 4 and 5, for each antibody pair, standard curves were determined on the antibody pairs in concentrations ranging from 5 ng/mL to 20 ng/mL. Of two top antibody pairs identified, both contained MM08 at the TL. Although the MM08-MM05 pair did not perform as well using LFA for virus detection, it performed well using ELISA.

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

[0093] Various pH conditions were tested for optimal conjugation of antibodies to gold nanoshells for LFA in order to increase assay sensitivity. 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.

Example 6: Optimizing antibody loading and EDC/Sulfo-NHS ratios

[0094] Antibody loading and EDC/Sulfo-NHS ratios were tested for optimal conjugation of antibodies to gold nanoshells for LFA in order to increase assay sensitivity. Conjugation optimization was performed with the following antibodies: R5, MM05, and 5B1. 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, recombinant N protein was added at a concentration of 10 ng/mL. In the negative sample, no recombinant N protein was added. 1:1 and 1:3 molar ratios of EDC to Sulfo-NHS was tested with antibody loading concentrations of 10, 20 and 40 pg/mL. As shown in Figure 7, an antibody loading concentration of 20 pg/mL and a 1 : 1 molar ratio of EDC to Sulfo-NHS showed significant signal-to-noise ratios at the TL and therefore are the determined optimal conditions for LFA.

Example 7: Testing ExonBio 3E6 paired with multiple antibodies

[0095] The ExonBio 3E6 antibody was tested with the recombinant N protein using LFA to identify target antibody pairs. In the positive sample, antibody pairs were tested with heat inactivated virus at a concentration of 10 ng/mL. In the negative sample, 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 8: Recombinant N protein titration

[0096] 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. As shown in Figures 9 and 10, MM08-3E6 and 5B1-3E6 showed high sensitivity to recombinant N protein binding, whereas MM08-R04 and 5B1-R04 showed low sensitivity.

Example 9: Characterization of MM08-3E6 and 5B1-3E6

[0097] 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). In the positive sample, recombinant N protein was immobilized at the TL at a concentration of 1 ng/mL. In the negative sample, no recombinant N protein was immobilized. As shown in Figure 11, 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

[0098] 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. As shown in Figure 12, 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

[0099] Various amounts of conjugated particles and sample volumes were evaluated for MM08-3E6 binding with recombinant N protein. 3E6-bound conjugate concentrations of 20, 30 and 40 optical density units (OD) were evaluated. In the positive sample, 1 ng/mL of recombinant N protein was added to the sample. In the negative sample, no recombinant N protein, but rather PBS buffer, was added. Three buffers, including PBS, Fantibody, and Tris, were each evaluated. As shown in Figure 13, 20 pL of the recombinant N protein at a concentration of 1 ng/mL and 35 pL of the buffer was loaded to the LFA test strip, which is loaded with the MM08-3E6 conjugate. As shown in Figure 14, 35 pL of the recombinant N protein and 20 pL of one of the three buffers was loaded to the LFA test strip, which is loaded with the MM08-3E6 conjugate. Referring to both Figures 13 and 14, signal intensity at the TL increased slightly when the OD of the MM08-3E6 conjugate was increased from 20 to 30 OD but changes were insignificant from 30 to 40 OD. Furthermore, increasing the sample volume increased signal intensity at the TL as well. An OD of 30 was determined to be the optimal condition.

Example 12: Clinical testing of antibody pairs with human saliva samples

[0100] 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. As shown in Figure 15, the LFA test strip (left) was loaded with the MM08-3E6 conjugate, and the LFA test strip (right) was loaded with MM08-R4. In the LFA test strip loaded with MM08-3E6, saliva samples 5, 6, 8, 9 and 10 were detected. In the LFA test strip loaded with MM08-R4, saliva samples 5, 6, 8 and 10 were detected. Since the antibody pair MM08-R4 had a detection rate of 50%, MM08-R4 was not selected for further testing. The antibody pair MM08-3E6 was selected for further testing.

Example 13: Qualification of various COVID-19 strains for MM08-3E6 binding

[0101] 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.

Table 6: Signal intensities at the TL of the LFA test strips of MM08-3E6 binding with three COVID-19 strains

Example 14: Determining the limit of detection (LoD) of recombinant antigen

[0102] The limit of detection was evaluated for various antibody pairs against recombinant N protein derived from different cell lines. Antibody pairs 3E6-Meridian 9548 (“3E6/48”), 3E6:Meridian 9547-9548 (“3E6:47/48”), and 3E6/MM08 were tested against recombinant N protein derived from HEK 293 cells (GTX) and Escherichia coli ( E . coli, M). 10 OD of 3E6, 2 mg/mL of 48, 10 OD of 47, and 1 mg/mL of MM08 was used. As shown in Figure 18, 3E6- MM08 was identified with the highest signal-to-noise ratio and as a good candidate for detecting the recombinant antigen.

Example 15: Determining the LoD of heat inactivated virus

[0103] The limit of detection was evaluated for heat inactivated Washington strain virus against recombinant N protein derived from different cell lines. Antibody pairs 3E6/48, 3E6:47/48, and 3E6/MM08 were tested against recombinant N protein derived from HEK 293 cells (GTX) and Escherichia coli ( E . coli, M). 10 OD of 3E6, 2 mg/mL of 48, 10 OD of 47, and 1 mg/mL of MM08 was used. A concentration of 1.15xl0 ⁷ TCID50/mL of the heat inactivated virus was used. As shown in Figure 19, 3E6-MM08 was identified with the highest signal-to- noise ratio and as a good candidate for detecting the recombinant antigen.

Example 16: Clinical testing of antibody pairs with human nasal swab samples

[0104] 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) and four Lee BioSolutions nasal swab samples (86, 53, 55, 54) were used in the evaluation. Although viral load (Ct) information was not provided for the Reprocell samples, the samples varied in viral load. Lee BioSolutions samples varied in viral load (Ct). The antibody combinations evaluated included 3E6/48, 3E6:47/48, and 3E6/MM08. For the antibody combination 3E6/48, 3E6 was loaded at the TL, and 48 was loaded as the conjugate. For the antibody combination 3E6:47/48, both 3E6 and 47 were loaded at the TL, and 48 was loaded as the conjugate. For the antibody combination 3E6/MM08, 3E6 was loaded at the TL, and MM08 was loaded as the conjugate. As shown in Figure 20, nasal swab samples 53 and 54 were detected using 3E6/48, nasal swab samples 14, 18, 20, 53 and 54 were detected using 3E6:47/48, and nasal swab samples 13, 14, 18, 20, 53, 55 and 54 were detected using 3E6:47/MM08. 3E6/48 had a detection rate of 25%, 3E6:47/48 had detection rate of 62.5%, and 3E6:47/MM08 had a detection rate of 87.5%.

Example 17: LFA cassette and sample volume study

[0105] 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. For the antibody combination 3E6/(MM08+48), 3E6 was loaded at the TL, and both MM08 and 48 were loaded as conjugates. For the antibody combination (3E6+47)/(MM08+48), 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. For the antibody combination (3E6+47)/48, both 3E6 and 47 were loaded at the TL, and 48 was loaded as a conjugate. As shown in Figure 21, 3E6/(MM08+48) had a detection rate of 66.7%, (3E6+47)/(MM08+48) had a detection rate of 100%, 3E6/MM08 had a detection rate of 50%, and (3E6+47)/48 had a detection rate of 100%. [0106] Sensitivity of antibody binding was increased in the LFA cassette compared to an LFA test strip. Sensitivity of antibody binding was also increased when increasing the antibody pair-containing sample from 25 pL to 50 pL.