HIGH THROUGHPUT SCREENING FOR MONOCLONAL ANTIBODY PAIRS

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U S C § 119(e) to United States Provisional Patent Application Number 63/049,178 entitled HIGH THROUGHPUT SCREENING FOR MONOCLONAL ANTIBODY PAIRS, filed July 8, 2020, the contents of which is hereby incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application.

[0003] This patent disclosure contains material that is subject to copyright protection.

The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

BACKGROUND OF THE INVENTION

[0004] A viral infection is the proliferation of a harmful virus inside a subject’s body. Viruses depend on host cells for their reproduction. Coronavirus (CoV) disease COVID-19 is a fast spreading worldwide pandemic caused by a coronavirus strain. Symptoms can appear 2-14 days after exposure and include fever, cough, and shortness of breath. Flavivirus strains cause widespread morbidity and mortality throughout the world. They are transmitted by ticks and are responsible of encephalitis and hemorrhagic diseases.

SUMMARY OF THE INVENTION

[0005] In certain aspects, the invention provides an immunoassay for testing the suitability of a monoclonal antibody pair for use in a lateral flow assay to detect a target protein, wherein the monoclonal antibody pair consists of a first monoclonal antibody and a second monoclonal antibody, wherein the first and the second monoclonal antibodies are capable of specifically binding the target protein. [0006] In some embodiments, the first and the second monoclonal antibodies have been purified with protein L or protein G resins. In some embodiments, the first monoclonal antibody is immobilized on a solid support, and the second monoclonal antibody is conjugated to a visualizing agent. In some embodiments, the visualizing agent-conjugated second monoclonal antibody and the target protein are allowed to bind to the immobilized first monoclonal antibody at the same time. In some embodiments, the solid support is a nitrocellulose membrane.

[0007] In some embodiments, the visualizing reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 600-700 nm. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 500 nm.

[0008] In some embodiments, the first monoclonal antibody is different from the second monoclonal antibody. In some embodiments, the first monoclonal antibody is the same as the second monoclonal antibody.

[0009] In some embodiments, the target protein is a viral antigen. In some embodiments, the viral antigen is a coronavirus (CoV) antigen. In some embodiments, the coronavirus (CoV) antigen is a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is SARS-CoV-2 Spike Protein (SARS-CoV- 2S). In some embodiments, the coronavirus (CoV) antigen is SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0010] In some embodiments, the coronavirus (CoV) antigen is a portion of a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS- CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a portion of a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Spike Protein (SARS-CoV-2S). In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0011] In some embodiments, the viral antigen is a flavivirus antigen. In some embodiments, the flavivirus antigen is a protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is flavivirus precursor membrane (PrM) protein.

[0012] In some embodiments, the flavivirus antigen is a portion of protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is a portion of flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is a portion of flavivirus precursor membrane (PrM) protein.

[0013] In certain aspects, a high throughput immunoassay includes a multitude of immunoassays as described above, wherein the multitude of immunoassays are conducted at the same time and in the same device.

[0014] In some embodiments, the first monoclonal antibodies of more than one immunoassays are immobilized on the same solid support. In some embodiments, the high throughput assay is conducted for selecting a suitable monoclonal antibody pair for use in a lateral flow assay to detect a target protein, and the selection is from a collection of monoclonal antibodies capable of specifically binding the target protein, wherein the selected monoclonal antibody pair consists of a first monoclonal antibody and a second monoclonal antibody.

[0015] In some embodiments, a multitude of combinations of two monoclonal antibodies from the collection of monoclonal antibodies capable of specifically binding the target protein are tested in the same high throughput immunoassay as the first and the second monoclonal antibody. In some embodiments, in some of the multitude of combinations the first monoclonal antibody is the same as the second monoclonal antibody.

[0016] In some embodiments, every combination of two monoclonal antibodies from the collection of monoclonal antibodies capable of specifically binding the target protein as the first and the second antibody, respectively, is tested in the same high throughput immunoassay. In some embodiments, in some of the combinations the first monoclonal antibody is the same as the second monoclonal antibody.

[0017] In certain aspects, a high throughput immunoassay device includes a commercially available multi-well plate, multiple test trips, a holder for the test strips, wherein the distal end of each test strip is immersed in one well of the multiple-well plate, and wherein each test strip comprises a solid support for immobilized proteins or peptides. [0018] In some embodiments, each test strip comprises a proximal wick portion and a distal dipstick portion, wherein the end of each dipstick portion is immersed in one well of the multi-well plate. In some embodiments, the dipstick portion comprises a test area, wherein the test area can provide support for one immobilized protein or peptide. In some embodiments, the dipstick portion comprises multiple test areas, wherein each test area can provide support for one immobilized protein or peptide. In some embodiments, the multiple test areas of the dipstick portion are arranged in a single vertical row along the vertical axis of the dipstick. In some embodiments, the dipstick is shaped as a string of overlapping diamonds, wherein each diamond shape provides one test area. In some embodiments, the dipstick comprises three overlapping diamond shapes.

[0019] In some embodiments, the dipstick has one or more flow splitters, wherein the flow splitters are gaps in the test strips parallel to the vertical axis of the dipstick, wherein each flow splitter divides the dipstick into two half-strip dipstick sections at the level of the flow splitter, and wherein the two half-strip dipstick sections are to the left and to the right of the flow splitter. In some embodiments, each half-strip dipstick section provides one test area. In some embodiments, at least some of the diamond shapes encompass a flow splitter and are divided into two half-diamond dipstick sections at the level of the flow splitter. In some embodiments, each half-diamond dipstick section provides one test area. In some embodiments, the wick portion of the test strip comprises a T-shaped handle of the test strip. [0020] In some embodiments, the holder for the test strips comprises a plate-shaped aligner with a multitude of slots, wherein the aligner is placed above the multi-well plate, wherein each slot can accommodate one test strip, and wherein the positions of the slots correspond to the positions of the wells in the multi-well plate so that the distal end of the test strip vertically extending through a slot can be localized in a well. In some embodiments, the aligner slots are longer than the width of the dipstick portion of the test strip but shorter than the width of the top of the T-shaped wick portion handle. In some embodiments, the width of the aligner slots is similar to the thickness of the test strips, and wherein the width of the aligner slots is sufficient to accommodate the test strip extending through the slot. In some embodiments, the aligner slots are diagonally oriented to the aligner.

[0021] In some embodiments, the holder for the test strips further comprises a top plate with a multitude of top plate slots, wherein the top plate is placed above the aligner, wherein each top plate slot can accommodate one test strip, and wherein the positions of the top plate slots align with the positions of the aligner slots below in diagonal orientations so that a test strip vertically extending through a top plate slot can also extend through an aligner slot below. In some embodiments, the top plate slots are longer than the width of the dipstick portion of the test strip but shorter than the width of the top of the T-shaped wick portion handle. In some embodiments, the width of the top plate slots is similar to the thickness of the test strips, and wherein the width of the top plate slots is sufficient to accommodate the test strip extending through the slot. In some embodiments, there is one top plate slot and one aligner slot that corresponds to every well in the multi-well plate.

[0022] In some embodiments, the high throughput immunoassay device further includes an outer housing container, wherein the multi-well plate, the test strips, the aligner, and the top plate are all placed in the housing container. In some embodiments, the multi-well plate is a 96-well plate. In some embodiments, the multi-well plate is a flat-bottom 96-well plate. In some embodiments, the multi-well plate is a round-bottom 96-well plate.

[0023] In some embodiments, there are snap tabs on the inner walls of the housing container. In some embodiments, the snap tabs can snap-fit to the multi-well plates. In some embodiments, the device can accommodate multiple commercially available multi-well plates and multiple holders for the test strips.

[0024] In certain aspects, the invention provides an immunoassay for testing the binding capacity between a monoclonal antibody and a protein or peptide, wherein the protein or peptide is immobilized on a solid support, and wherein the monoclonal antibody is conjugated to a visualizing agent.

[0025] In some embodiments, the visualizing agent -conjugated monoclonal antibody is allowed to bind to the immobilized protein or peptide. In some embodiments, the solid support is a nitrocellulose membrane. In some embodiments, the visualizing reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 600-700 nm. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 500 nm. [0026] In some embodiments, the protein or peptide is a viral antigen. In some embodiments, the protein or peptide is a portion of a viral antigen. In some embodiments, the portion of a viral antigen has a length of 9-15 amino acid.

[0027] In some embodiments, the viral antigen is a coronavirus (CoV) antigen. In some embodiments, the coronavirus (CoV) antigen is a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is SARS-CoV-2 Spike Protein (SARS-CoV-2S). In some embodiments, the coronavirus (CoV) antigen is SARS- CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0028] In some embodiments, the viral antigen is a flavivirus antigen. In some embodiments, the flavivirus antigen is a protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is flavivirus precursor membrane (PrM) protein.

[0029] In certain aspects, the invention provides a high throughput immunoassay which includes a multitude of immunoassays previously described, wherein the multitude of the immunoassays are conducted at the same time and in the same device.

[0030] In some embodiments, the proteins or peptides of more than one immunoassays are immobilized on the same solid support. In some embodiments, the high throughput immunoassay is conducted to map the peptide recognized by a monoclonal antibody, wherein the peptide is localized within a target protein recognized by the monoclonal antibody. In some embodiments, a multitude of peptides from the target protein are tested in the same high throughput immunoassay against the same monoclonal antibody, wherein the protein is known to bind to the antibody.

[0031] In some embodiments, the target protein is a viral antigen. In some embodiments, the target protein is a coronavirus (CoV) antigen. In some embodiments, the coronavirus (CoV) antigen is a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is SARS-CoV-2 Spike Protein (SARS-CoV- 2S). In some embodiments, the coronavirus (CoV) antigen is SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0032] In some embodiments, the coronavirus (CoV) antigen is a portion of a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS- CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a portion of a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Spike Protein (SARS-CoV-2S). In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0033] In some embodiments the viral antigen is a flavivirus antigen. In some embodiments, the flavivirus antigen is a protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is flavivirus precursor membrane (PrM) protein.

[0034] In some embodiments, the flavivirus antigen is a portion of protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is a portion of flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is a portion of flavivirus precursor membrane (PrM) protein.

[0035] In certain aspects, the invention provides a high throughput screening method for selecting a suitable monoclonal antibody pair consisting of a first and a second monoclonal antibody for use in a lateral flow assay to detect a target protein, the method comprising: obtaining a collection of candidate monoclonal antibodies that specifically bind to the target protein; providing a high throughput immunoassay device of any one of the claims 35-58; providing at least one immunoassay test strip secured within the high throughput immunoassay device; applying at least one candidate monoclonal antibody on at least one of the test areas of the at least one test strip, except the uppermost test area of each of the at least one test strips; applying a control antibody in the uppermost test area of each of the at least one test strip; conjugating a visualizing agent to at least one candidate monoclonal antibody; applying the target protein and the at least one visualizing agent conjugated candidate monoclonal antibody in the multi-well plate, wherein each well is provided with the target protein and one conjugated candidate monoclonal antibody, and wherein the positions of the wells correspond to the positions of the at least one test strips; immersing the distal end of the at least one test strips to the corresponding wells with the target protein and the at least one visualizing agent conjugated-candidate monoclonal antibody; allowing the target protein and the at least one visualizing agent-conjugated candidate monoclonal antibody to vertically defuse through the test strip and contact the immobilized monoclonal antibodies and the control antibody; incubating the reaction for at least 15-30 minutes; and recoding the signal of each test area on the at least one test strip.

[0036] In some embodiments, the control antibody is an antibody that specifically binds the candidate monoclonal antibodies. In some embodiments, the control antibody is an antibody that specifically binds the Fc portion of the candidate monoclonal antibodies.

[0037] In some embodiments, each candidate monoclonal antibody is conjugated with a visualization agent and tested against each candidate monoclonal antibody immobilized on the test strip. In some embodiments, each candidate monoclonal antibody is immobilized on at least one test strip and tested against each candidate monoclonal antibody conjugated with a visualization agent. In some embodiments, each candidate monoclonal antibody is conjugated with a visualizing agent and tested against the same candidate monoclonal antibody on a test strip.

[0038] In some embodiments, the portion of the test strip that contacts the immobilized antibodies is nitrocellulose membrane. In some embodiments, the visualizing reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 600-700 nm. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 500 nm. [0039] In some embodiments, the target protein is a viral antigen. In some embodiments, the viral antigen is a coronavirus (CoV) antigen. In some embodiments, the coronavirus (CoV) antigen is a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is SARS-CoV-2 Spike Protein (SARS-CoV- 2S). In some embodiments, the coronavirus (CoV) antigen is SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0040] In some embodiments, the coronavirus (CoV) antigen is a portion of a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS- CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a portion of a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Spike Protein (SARS-CoV-2S). In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0041] In some embodiments, the viral antigen is a flavivirus antigen. In some embodiments, the flavivirus antigen is a protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is flavivirus precursor membrane (PrM) protein.

[0042] In some embodiments, the flavivirus antigen is a portion of protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1),

Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is a portion of flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is a portion of flavivirus precursor membrane (PrM) protein. [0043] In certain aspects, the invention provides a high throughput selection platform for selecting a suitable monoclonal antibody pair consisting of a first and a second monoclonal antibody for use in a lateral flow assay to detect a target protein, wherein detecting the target protein comprises distinguishing the target protein from non-target proteins that are similar to the target protein, the method including: conducting the high throughput screening method previously described with the target protein and the collection of candidate monoclonal antibodies capable of binding the target protein; conducting the high throughput screening method previously described with each of the non-target proteins that are similar to the target protein and the collection of candidate monoclonal antibodies capable of binding the target protein; comparing the signal intensity of the tested antibody pairings for the target protein and for each of the non-target proteins that are similar to the target protein; selecting the antibody parings that are capable of recognizing the target protein and distinguishing between the target protein and the non-target proteins that are similar to the target protein.

[0044] In some embodiments, the high throughput screening with the non-target proteins similar to the target protein and the high throughput screening with the target protein are conducted in the same device and at the same time.

[0045] In some embodiments, the target protein is a viral antigen. In some embodiments, the target protein viral antigen is a coronavirus (CoV) antigen. In some embodiments, the target protein coronavirus (CoV) antigen is a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a protein from SARS-CoV-2. In some embodiments, the target protein coronavirus (CoV) antigen is the SARS-CoV-2 Spike Protein (SARS-CoV-2S). In some embodiments, the target protein coronavirus (CoV) antigen is the SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N). [0046] In some embodiments, the target protein coronavirus (CoV) antigen is a portion of a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV- 2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the target protein coronavirus (CoV) antigen is a portion of a protein from SARS-CoV-2. In some embodiments, the target protein coronavirus (CoV) antigen is a portion of SARS-CoV- 2 Spike Protein (SARS-CoV-2S). In some embodiments, the target protein coronavirus (CoV) antigen is a portion of SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0047] In some embodiments, the non-target proteins that are similar to the target protein are viral antigens. In some embodiments, the non-target protein viral antigens are coronavirus (CoV) antigens. In some embodiments, at least some of the non-target protein coronavirus (CoV) antigens are proteins from coronavirus (CoV) strains selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, at least some of the non-target protein coronavirus (CoV) antigens are proteins from SARS-CoV-2. In some embodiments, at least some of the non target protein coronavirus (CoV) antigens are not proteins from SARS-CoV-2. In some embodiments, at least some of the non-target protein coronavirus (CoV) antigens are Spike Protein (S) from a coronavirus (CoV) strains selected from the group consisting of SARS- CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, at least some of the coronavirus (CoV) antigens are Nucleocapsid protein (N) from a coronavirus (CoV) strains selected from the group consisting of SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU 1.

[0048] In some embodiments, at least some of the non-target protein coronavirus (CoV) antigens are portions of a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, at least some of the non-target protein coronavirus (CoV) antigens are portions of proteins from SARS-CoV-2. In some embodiments, at least some of the non target protein coronavirus (CoV) antigens are not portions of proteins from SARS-CoV-2. In some embodiments, at least some of the non-target protein coronavirus (CoV) antigens are portions from Spike Protein (S) from a coronavirus (CoV) strains selected from the group consisting of SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, at least some of the non-target protein coronavirus (CoV) antigens are portions from Nucleocapsid protein (N) from a coronavirus (CoV) strains selected from the group consisting of SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1.

[0049] In some embodiments, the target protein viral antigen is a flavivirus antigen. In some embodiments, the target protein flavivirus antigen is a protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the target protein flavivirus antigen is flavivirus non- structural protein 1 (NS1). In some embodiments, the target protein flavivirus antigen is flavivirus precursor membrane (PrM) protein.

[0050] In some embodiments, the target protein flavivirus antigen is a portion of protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the target protein flavivirus antigen is a portion of flavivirus non- structural protein 1 (NS1). In some embodiments, the target protein flavivirus antigen is a portion of flavivirus precursor membrane (PrM) protein.

[0051] In some embodiments, the non-target protein viral antigens are flavivirus antigens. In some embodiments, at least some of the non-target protein flavivirus antigens are proteins from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, at least some of the non-target protein flavivirus antigens are flavivirus non- structural protein 1 (NS1). In some embodiments, at least some of the non-target protein flavivirus antigens are flavivirus precursor membrane (PrM) protein.

[0052] In some embodiments, at least some of the non-target protein flavivirus antigens are portions of protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, at least some of the non-target protein flavivirus antigens are portions of flavivirus non- structural protein 1 (NS1). In some embodiments, at least some of the non-target protein flavivirus antigens are portions of flavivirus precursor membrane (PrM) protein. [0053] In certain aspects, the invention provides a high throughput screening method for mapping the peptide that is recognized by a monoclonal antibody within a target protein recognized by the monoclonal antibody, the method including: obtaining a collection of overlapping candidate peptides that are fragments of the protein; providing a high throughput immunoassay device of any one of the claims 35-58; providing at least one immunoassay test strip secured within the high throughput immunoassay device; applying at least one candidate peptide on at least one of the test areas of the at least one test strip, except the uppermost test areas of each of the at least one test strips; applying a control antibody in the uppermost test area of each of the at least one test strip; conjugating a visualizing agent to the monoclonal antibody; applying the visualizing agent conjugated monoclonal antibody in wells of the multi-well plate corresponding the positions of the at least one test strips; immersing the distal end of the at least one test strips into the corresponding wells with the visualizing agent conjugated monoclonal antibody; allowing the visualizing agent conjugated monoclonal antibody to vertically defuse through the test strips and contact the immobilized peptides and the control antibody; incubating the reaction for at least 15-30 minutes; recoding the signal of each test area on the at least one test strip.

[0054] In some embodiments, the control antibody is an antibody that specifically binds the monoclonal antibody. In some embodiments, the control antibody is an antibody against the Fc portion of the monoclonal antibody. In some embodiments, each of the candidate peptides is immobilized on at least one test strip and tested against the visualizing agent conjugated monoclonal antibody.

[0055] In some embodiments, the portion of the test strip that contacts the immobilized candidate peptides is nitrocellulose membrane. In some embodiments, the visualizing reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 600-700 nm. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 500 nm.

[0056] In some embodiments, the target protein is a viral antigen. In some embodiments, the viral antigen is a coronavirus (CoV) antigen. In some embodiments, the coronavirus (CoV) antigen is a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is the SARS-CoV-2 Spike Protein (SARS-CoV- 2S). In some embodiments, the coronavirus (CoV) antigen is the SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0057] In some embodiments, the coronavirus (CoV) antigen is a portion of a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS- CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a portion of protein from SARS-CoV-2 In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Spike Protein (SARS-CoV-2S). In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0058] In some embodiments, the viral antigen is a flavivirus antigen. In some embodiments, the flavivirus antigen is a protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is flavivirus precursor membrane (PrM) protein.

[0059] In some embodiments, the flavivirus antigen is a portion of protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1),

Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is a portion of flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is a portion of flavivirus precursor membrane (PrM) protein.

[0060] In some embodiments, the invention provides a method to utilize the high throughput immunoassay device as described above, wherein one group of test strips are utilized to conduct the high throughput screening for antibody paring as described above, and another group of test strips are utilized to conduct the high throughput mapping as described above. [0061] In some embodiments, the tests utilizing the two groups of test strips are started and interpreted at the same time. In some embodiments, the tests utilizing the two groups of test strips are started or interpreted at different times.

BRIEF DESCRIPTION OF FIGURES

[0062] The patent or application fde contains at least one drawing originally in color. [0063] Figure 1 shows a schematic diagram of some embodiments of individual components of a high throughout immunoassay device.

[0064] Figure 2 shows a photo of one embodiment of a high-throughput immunoassay device.

[0065] Figure 3 shows detailed technical specification of one embodiment of Test Strip Assembly, Dipstick, and Wick of the high-throughput immunoassay device.

[0066] Figure 4 shows detailed technical specification of one embodiment of the Top Plate high-throughput immunoassay device.

[0067] Figure 5 shows detailed technical specification of one embodiment of the Divider/ Aligner high-throughput immunoassay device.

[0068] Figure 6 shows detailed technical specification of embodiments of Holders of the high-throughput immunoassay device for 96-well round bottom and flat bottom plates, respectively.

[0069] Figure 7 shows a high throughput screen test to identify monoclonal antibody pairing to detect specific viral targets. In the left panel, the test areas are loaded with the immobilized antibodies a-mouse Fc (control antibody), a-SARS-CoV-2 S, and a-SARS- CoV-2 N. The antibodies a-SARS-CoV-2 S and a-SARS-CoV-2 S are also conjugated to nanoparticles. The peptides SARS-CoV-2 S and N are introduced to the reactions as well. In the right panel, the test areas are (left to the splitters): a-human Fc (control antibody), a- DENV-2 NS1, and a-SARS-CoV-2 S The test areas right to the splitters are a-mouse Fc (control antibody), a-DENV-3 NS1, and a-SARS-CoV-2 N. The conjugated antibodies are a-DENV-2 NS1, a-DENV-3 NS1, a-SARS-CoV-2 S, and a-SARS-CoV-2 N. The antigens introduced to the reactions are DENV-2 NS1, SARS-CoV-2 S, and SARS-CoV-2 N.

[0070] Figure 8 shows a high throughput screen that identifies monoclonal antibody pairs that detect SARS-Coronaviruses with varying levels of specificity and strength.

[0071] Figure 9 shows a high throughput screen to map protein peptides recognized by monoclonal antibodies. Four peptides corresponding to different portions ofDEV S-1 were immobilized on the dipstick test areas. The upper right rest area was loaded with a control antibody a-mouse Fc. Conjugated antibody a-DENV NS 1 was allowed to bind to the immobilized peptides and control antibody.

[0072] Figure 10 shows Leinco antibody grid from a high throughput screening.

[0073] Figure 11 shows stepwise strategy for identifying mAbs that differentiate the closely related NS1 proteins of DENV1-4 and ZIKV. Step 1 : mouse hybridomas are generated by immunizing mice with viral NS1 protein antigen, and the resulting hybridomas are screened using ELISA against multiple recombinant NS1 proteins. Step 2: hybridoma cell supernatants containing monoclonal antibodies are screened using flow cytometry for recognition of native NS1 proteins released by Yero cells infected with DENV serotypes or ZIKV. Step 3 : purified monoclonal antibodies are tested in pairs for specific recognition of DENV or ZIKV NS1 proteins in immunochromatography rapid tests that run in about 30 minutes. Step 4: linear epitope mapping is performed to identify NS1 sites recognized by monoclonal antibodies used in the rapid tests. Step 5: patient serum from DENV- or ZIKV- infected patients is tested using the immunochromatography dipsticks. Step 6: images of test results are captured using image recognition on a mobile phone camera, and analyzed objectively to quantify signals.

[0074] Figure 12 shows Repertoire of antigen-specific antibodies among DENV and ZIKV mAbs. The pie charts show the percentages of dengue serotype-specific or Zika virus- specific monoclonal antibodies as defined during screening. The values were determined by screening the hybridomas against the antigens labeled on the figures. The ZIKA virus monoclonal antibody preparation was done by two methods (splenic B cells and lymph node tissue).

[0075] Figure 13 shows Matrix of mAb pair trials. To define functional antibody pairs, one antibody was conjugated to gold nanoparticles, and one antibody was adsorbed to nitrocellulose membrane. The resulting nanoparticle conjugates-membrane pairs were tested using the specific serotype recombinant NS 1 protein (on the left axis: mock, DENV1, DENV2, DENV3, DENV 4, ZIKV, or a DV 1-4 mix of all four NS 1 proteins; highlighted in blue color). These proteins were present at a concentration of 1.6 ug/ml in the testing. Because of the large number of tests performed, the strips were analyzed and normalized in groups corresponding to nanoparticle lots and antibody dilutions. The signal for each test strip was quantified using Image! Background signal intensity was subtracted and the signal of greatest intensity was set at a value of “1”. For remaining strips, background was subtracted from signal intensity to yield a corrected intensity value. The corrected intensity value was then divided by the maximal signal value to yield a value between 0 and 1 that was plotted above to generate a relative binding affinity map. Dark green color: strong visual signal in the rapid test; light green color: weak visual signal in the rapid test; gray color: no signal in the rapid test. These tests were performed using lab-made gold nanoparticles, which perform equivalently in comparison to commercial Innova particles. The antibody pairs were tested at a sample concentration of 3 rg/ml of recombinant NS 1 protein. Left panel: dengue virus antibodies; Right panel: Zika virus antibodies, with the exception of 136, a dengue NS1 antibody that binds Zika virus NS1 with high affinity.

[0076] Figure 14 shows NS1 protein alignment and linear epitope mapping of the 10 antibodies used to run the DENV serotype-specific NS1 rapid tests, pan-DENVNSl test, and ZIKVNS1 test. (A) Table listing mAb names, mAb immunochromatography applications, mAb linear epitope sequences and starting amino acid positions, and NS1 domain positions. (B) Comparison of amino acid similarity based on analysis ofNSl protein sequences from the following viruses: DENV1 (strain Singapore/S275/1990), accession number P33478; DENV2 (strain New Guinea C), accession number AAA42941; DENV3 (strain Philippines/H87/1956), accession number AAA99437; DENV4 (strain Singapore/8976/1995), accession number AAV31422; ZIKV, accession number KU497555.1. Amino acid sequences were compared using Color Align Conservation www.bioinformatics.org/sms2/color_align_cons.html to enhance the output of sequence alignment program. Residues that are identical among the sequences are boxed. Linear peptide epitopes (B) are italicized and indicated in color in the figure, with the key to the right of the figure.

DETAILED DESCRIPTION OF THE INVENTION

[0077] Because ZD V and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. Such cross-reactions and false-positive results can also occur for related coronaviruses. There is a need for characterization of monoclonal antibody pairs that can be translated into rapid immunochromatography tests to specifically detect the viral protein antigens and distinguish between the closely related viruses and serotypes without cross-reaction, to enable a rapid and reliable test for viral infections such as the coronavirus strain SARS-CoV-2.

[0078] Non-Limiting Embodiments of the Subject Matter [0079] In certain aspects, the invention provides an immunoassay for testing the suitability of a monoclonal antibody pair for use in a lateral flow assay to detect a target protein, wherein the monoclonal antibody pair consists of a first monoclonal antibody and a second monoclonal antibody, wherein the first and the second monoclonal antibodies are capable of specifically binding the target protein.

[0080] In some embodiments, the first and the second monoclonal antibodies have been purified with protein L or protein G resins. In some embodiments, the first monoclonal antibody is immobilized on a solid support, and the second monoclonal antibody is conjugated to a visualizing agent. In some embodiments, the visualizing agent-conjugated second monoclonal antibody and the target protein are allowed to bind to the immobilized first monoclonal antibody at the same time. In some embodiments, the solid support is a nitrocellulose membrane.

[0081] In some embodiments, the visualizing reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 600-700 nm. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 500 nm.

[0082] In some embodiments, the first monoclonal antibody is different from the second monoclonal antibody. In some embodiments, the first monoclonal antibody is the same as the second monoclonal antibody.

[0083] In some embodiments, the target protein is a viral antigen. In some embodiments, the viral antigen is a coronavirus (CoV) antigen. In some embodiments, the coronavirus (CoV) antigen is a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is SARS-CoV-2 Spike Protein (SARS-CoV- 2S). In some embodiments, the coronavirus (CoV) antigen is SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0084] In some embodiments, the coronavirus (CoV) antigen is a portion of a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS- CoV, MERS-CoV, 229E, NL63, OC43, and HKUE In some embodiments, the coronavirus (CoV) antigen is a portion of a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Spike Protein (SARS-CoV-2S) In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0085] In some embodiments, the viral antigen is a flavivirus antigen. In some embodiments, the flavivirus antigen is a protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is flavivirus precursor membrane (PrM) protein.

[0086] In some embodiments, the flavivirus antigen is a portion of protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is a portion of flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is a portion of flavivirus precursor membrane (PrM) protein.

[0087] In certain aspects, a high throughput immunoassay includes a multitude of immunoassays as described above, wherein the multitude of immunoassays are conducted at the same time and in the same device.

[0088] In some embodiments, the first monoclonal antibodies of more than one immunoassays are immobilized on the same solid support. In some embodiments, the high throughput assay is conducted for selecting a suitable monoclonal antibody pair for use in a lateral flow assay to detect a target protein, and the selection is from a collection of monoclonal antibodies capable of specifically binding the target protein, wherein the selected monoclonal antibody pair consists of a first monoclonal antibody and a second monoclonal antibody. [0089] In some embodiments, a multitude of combinations of two monoclonal antibodies from the collection of monoclonal antibodies capable of specifically binding the target protein are tested in the same high throughput immunoassay as the first and the second monoclonal antibody. In some embodiments, in some of the multitude of combinations the first monoclonal antibody is the same as the second monoclonal antibody.

[0090] In some embodiments, every combination of two monoclonal antibodies from the collection of monoclonal antibodies capable of specifically binding the target protein as the first and the second antibody, respectively, is tested in the same high throughput immunoassay. In some embodiments, in some of the combinations the first monoclonal antibody is the same as the second monoclonal antibody.

[0091] In certain aspects, a high throughput immunoassay device includes a commercially available multi-well plate, multiple test trips, a holder for the test strips, wherein the distal end of each test strip is immersed in one well of the multiple-well plate, and wherein each test strip comprises a solid support for immobilized proteins or peptides. [0092] In some embodiments, each test strip comprises a proximal wick portion and a distal dipstick portion, wherein the end of each dipstick portion is immersed in one well of the multi-well plate. In some embodiments, the dipstick portion comprises a test area, wherein the test area can provide support for one immobilized protein or peptide. In some embodiments, the dipstick portion comprises multiple test areas, wherein each test area can provide support for one immobilized protein or peptide. In some embodiments, the multiple test areas of the dipstick portion are arranged in a single vertical row along the vertical axis of the dipstick. In some embodiments, the dipstick is shaped as a string of overlapping diamonds, wherein each diamond shape provides one test area. In some embodiments, the dipstick comprises three overlapping diamond shapes.

[0093] In some embodiments, the dipstick has one or more flow splitters, wherein the flow splitters are gaps in the test strips parallel to the vertical axis of the dipstick, wherein each flow splitter divides the dipstick into two half-strip dipstick sections at the level of the flow splitter, and wherein the two half-strip dipstick sections are to the left and to the right of the flow splitter. In some embodiments, each half-strip dipstick section provides one test area. In some embodiments, at least some of the diamond shapes encompass a flow splitter and are divided into two half-diamond dipstick sections at the level of the flow splitter. In some embodiments, each half-diamond dipstick section provides one test area. In some embodiments, the wick portion of the test strip comprises a T-shaped handle of the test strip. [0094] In some embodiments, the holder for the test strips comprises a plate-shaped aligner with a multitude of slots, wherein the aligner is placed above the multi-well plate, wherein each slot can accommodate one test strip, and wherein the positions of the slots correspond to the positions of the wells in the multi-well plate so that the distal end of the test strip vertically extending through a slot can be localized in a well. In some embodiments, the aligner slots are longer than the width of the dipstick portion of the test strip but shorter than the width of the top of the T-shaped wick portion handle. In some embodiments, the width of the aligner slots is similar to the thickness of the test strips, and wherein the width of the aligner slots is sufficient to accommodate the test strip extending through the slot. In some embodiments, the aligner slots are diagonally oriented to the aligner.

[0095] In some embodiments, the holder for the test strips further comprises a top plate with a multitude of top plate slots, wherein the top plate is placed above the aligner, wherein each top plate slot can accommodate one test strip, and wherein the positions of the top plate slots align with the positions of the aligner slots below in diagonal orientations so that a test strip vertically extending through a top plate slot can also extend through an aligner slot below. In some embodiments, the top plate slots are longer than the width of the dipstick portion of the test strip but shorter than the width of the top of the T-shaped wick portion handle. In some embodiments, the width of the top plate slots is similar to the thickness of the test strips, and wherein the width of the top plate slots is sufficient to accommodate the test strip extending through the slot. In some embodiments, there is one top plate slot and one aligner slot that corresponds to every well in the multi-well plate.

[0096] In some embodiments, the high throughput immunoassay device further includes an outer housing container, wherein the multi-well plate, the test strips, the aligner, and the top plate are all placed in the housing container. In some embodiments, the multi-well plate is a 96-well plate. In some embodiments, the multi-well plate is a flat-bottom 96-well plate. In some embodiments, the multi-well plate is a round-bottom 96-well plate.

[0097] In some embodiments, there are snap tabs on the inner walls of the housing container. In some embodiments, the snap tabs can snap-fit to the multi-well plates. In some embodiments, the device can accommodate multiple commercially available multi-well plates and multiple holders for the test strips.

[0098] In certain aspects, the invention provides an immunoassay for testing the binding capacity between a monoclonal antibody and a protein or peptide, wherein the protein or peptide is immobilized on a solid support, and wherein the monoclonal antibody is conjugated to a visualizing agent.

[0099] In some embodiments, the visualizing agent -conjugated monoclonal antibody is allowed to bind to the immobilized protein or peptide. In some embodiments, the solid support is a nitrocellulose membrane. In some embodiments, the visualizing reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 600-700 nm. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 500 nm.

[0100] In some embodiments, the protein or peptide is a viral antigen. In some embodiments, the protein or peptide is a portion of a viral antigen. In some embodiments, the portion of a viral antigen has a length of 9-15 amino acid.

[0101] In some embodiments, the viral antigen is a coronavirus (CoV) antigen. In some embodiments, the coronavirus (CoV) antigen is a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is SARS-CoV-2 Spike Protein (SARS-CoV-2S). In some embodiments, the coronavirus (CoV) antigen is SARS- CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0102] In some embodiments, the viral antigen is a flavivirus antigen. In some embodiments, the flavivirus antigen is a protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is flavivirus precursor membrane (PrM) protein.

[0103] In certain aspects, the invention provides a high throughput immunoassay which includes a multitude of immunoassays previously described, wherein the multitude of the immunoassays are conducted at the same time and in the same device.

[0104] In some embodiments, the proteins or peptides of more than one immunoassays are immobilized on the same solid support. In some embodiments, the high throughput immunoassay is conducted to map the peptide recognized by a monoclonal antibody, wherein the peptide is localized within a target protein recognized by the monoclonal antibody. In some embodiments, a multitude of peptides from the target protein are tested in the same high throughput immunoassay against the same monoclonal antibody, wherein the protein is known to bind to the antibody.

[0105] In some embodiments, the target protein is a viral antigen. In some embodiments, the target protein is a coronavirus (CoV) antigen. In some embodiments, the coronavirus (CoV) antigen is a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is SARS-CoV-2 Spike Protein (SARS-CoV- 2S). In some embodiments, the coronavirus (CoV) antigen is SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0106] In some embodiments, the coronavirus (CoV) antigen is a portion of a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS- CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a portion of a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Spike Protein (SARS-CoV-2S). In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0107] In some embodiments the viral antigen is a flavivirus antigen. In some embodiments, the flavivirus antigen is a protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is flavivirus precursor membrane (PrM) protein.

[0108] In some embodiments, the flavivirus antigen is a portion of protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1),

Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) vims, West Nile vims (WNV), Deer tick (DTV) tick-borne encephalitis vims (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever vims (OE1FV). In some embodiments, the flavivirus antigen is a portion of flavivirus non-stmctural protein 1 (NS1). In some embodiments, the flavivirus antigen is a portion of flavivirus precursor membrane (PrM) protein.

[0109] In certain aspects, the invention provides a high throughput screening method for selecting a suitable monoclonal antibody pair consisting of a first and a second monoclonal antibody for use in a lateral flow assay to detect a target protein, the method comprising: obtaining a collection of candidate monoclonal antibodies that specifically bind to the target protein; providing a high throughput immunoassay device of any one of the claims 35-58; providing at least one immunoassay test strip secured within the high throughput immunoassay device; applying at least one candidate monoclonal antibody on at least one of the test areas of the at least one test strip, except the uppermost test area of each of the at least one test strips; applying a control antibody in the uppermost test area of each of the at least one test strip; conjugating a visualizing agent to at least one candidate monoclonal antibody; applying the target protein and the at least one visualizing agent conjugated candidate monoclonal antibody in the multi-well plate, wherein each well is provided with the target protein and one conjugated candidate monoclonal antibody, and wherein the positions of the wells correspond to the positions of the at least one test strips; immersing the distal end of the at least one test strips to the corresponding wells with the target protein and the at least one visualizing agent conjugated-candidate monoclonal antibody; allowing the target protein and the at least one visualizing agent-conjugated candidate monoclonal antibody to vertically defuse through the test strip and contact the immobilized monoclonal antibodies and the control antibody; incubating the reaction for at least 15-30 minutes; and recoding the signal of each test area on the at least one test strip.

[0110] In some embodiments, the control antibody is an antibody that specifically binds the candidate monoclonal antibodies. In some embodiments, the control antibody is an antibody that specifically binds the Fc portion of the candidate monoclonal antibodies.

[0111] In some embodiments, each candidate monoclonal antibody is conjugated with a visualization agent and tested against each candidate monoclonal antibody immobilized on the test strip. In some embodiments, each candidate monoclonal antibody is immobilized on at least one test strip and tested against each candidate monoclonal antibody conjugated with a visualization agent. In some embodiments, each candidate monoclonal antibody is conjugated with a visualizing agent and tested against the same candidate monoclonal antibody on a test strip.

[0112] In some embodiments, the portion of the test strip that contacts the immobilized antibodies is nitrocellulose membrane. In some embodiments, the visualizing reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 600-700 nm. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 500 nm.

[0113] In some embodiments, the target protein is a viral antigen. In some embodiments, the viral antigen is a coronavirus (CoV) antigen. In some embodiments, the coronavirus (CoV) antigen is a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is SARS-CoV-2 Spike Protein (SARS-CoV- 2S). In some embodiments, the coronavirus (CoV) antigen is SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0114] In some embodiments, the coronavirus (CoV) antigen is a portion of a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS- CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a portion of a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Spike Protein (SARS-CoV-2S). In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0115] In some embodiments, the viral antigen is a flavivirus antigen. In some embodiments, the flavivirus antigen is a protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is flavivirus precursor membrane (PrM) protein. [0116] In some embodiments, the flavivirus antigen is a portion of protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is a portion of flavivirus non- structural protein 1 (NS1). In some embodiments, the flavivirus antigen is a portion of flavivirus precursor membrane (PrM) protein.

[0117] In certain aspects, the invention provides a high throughput selection platform for selecting a suitable monoclonal antibody pair consisting of a first and a second monoclonal antibody for use in a lateral flow assay to detect a target protein, wherein detecting the target protein comprises distinguishing the target protein from non-target proteins that are similar to the target protein, the method including: conducting the high throughput screening method previously described with the target protein and the collection of candidate monoclonal antibodies capable of binding the target protein; conducting the high throughput screening method previously described with each of the non-target proteins that are similar to the target protein and the collection of candidate monoclonal antibodies capable of binding the target protein; comparing the signal intensity of the tested antibody pairings for the target protein and for each of the non-target proteins that are similar to the target protein; selecting the antibody parings that are capable of recognizing the target protein and distinguishing between the target protein and the non-target proteins that are similar to the target protein.

[0118] In some embodiments, the high throughput screening with the non-target proteins similar to the target protein and the high throughput screening with the target protein are conducted in the same device and at the same time.

[0119] In some embodiments, the target protein is a viral antigen. In some embodiments, the target protein viral antigen is a coronavirus (CoV) antigen. In some embodiments, the target protein coronavirus (CoV) antigen is a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a protein from SARS-CoV-2. In some embodiments, the target protein coronavirus (CoV) antigen is the SARS-CoV-2 Spike Protein (SARS-CoV-2S). In some embodiments, the target protein coronavirus (CoV) antigen is the SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N). [0120] In some embodiments, the target protein coronavirus (CoV) antigen is a portion of a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV- 2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the target protein coronavirus (CoV) antigen is a portion of a protein from SARS-CoV-2. In some embodiments, the target protein coronavirus (CoV) antigen is a portion of SARS-CoV- 2 Spike Protein (SARS-CoV-2S). In some embodiments, the target protein coronavirus (CoV) antigen is a portion of SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0121] In some embodiments, the non-target proteins that are similar to the target protein are viral antigens. In some embodiments, the non-target protein viral antigens are coronavirus (CoV) antigens. In some embodiments, at least some of the non-target protein coronavirus (CoV) antigens are proteins from coronavirus (CoV) strains selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, at least some of the non-target protein coronavirus (CoV) antigens are proteins from SARS-CoV-2. In some embodiments, at least some of the non target protein coronavirus (CoV) antigens are not proteins from SARS-CoV-2. In some embodiments, at least some of the non-target protein coronavirus (CoV) antigens are Spike Protein (S) from a coronavirus (CoV) strains selected from the group consisting of SARS- CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, at least some of the coronavirus (CoV) antigens are Nucleocapsid protein (N) from a coronavirus (CoV) strains selected from the group consisting of SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU 1.

[0122] In some embodiments, at least some of the non-target protein coronavirus (CoV) antigens are portions of a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, at least some of the non-target protein coronavirus (CoV) antigens are portions of proteins from SARS-CoV-2. In some embodiments, at least some of the non target protein coronavirus (CoV) antigens are not portions of proteins from SARS-CoV-2. In some embodiments, at least some of the non-target protein coronavirus (CoV) antigens are portions from Spike Protein (S) from a coronavirus (CoV) strains selected from the group consisting of SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, at least some of the non-target protein coronavirus (CoV) antigens are portions from Nucleocapsid protein (N) from a coronavirus (CoV) strains selected from the group consisting of SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. [0123] In some embodiments, the target protein viral antigen is a flavivirus antigen. In some embodiments, the target protein flavivirus antigen is a protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the target protein flavivirus antigen is flavivirus non- structural protein 1 (NS1). In some embodiments, the target protein flavivirus antigen is flavivirus precursor membrane (PrM) protein.

[0124] In some embodiments, the target protein flavivirus antigen is a portion of protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the target protein flavivirus antigen is a portion of flavivirus non- structural protein 1 (NS1). In some embodiments, the target protein flavivirus antigen is a portion of flavivirus precursor membrane (PrM) protein.

[0125] In some embodiments, the non-target protein viral antigens are flavivirus antigens. In some embodiments, at least some of the non-target protein flavivirus antigens are proteins from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, at least some of the non-target protein flavivirus antigens are flavivirus non- structural protein 1 (NS1). In some embodiments, at least some of the non-target protein flavivirus antigens are flavivirus precursor membrane (PrM) protein.

[0126] In some embodiments, at least some of the non-target protein flavivirus antigens are portions of protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue vims serotype 4 (DV4), Zika vims (ZIKV), Yellow Fever (YFV), Ilheus Vims (ILHV), Powassan (POWA), Japanese encephalitis (JE) vims, West Nile vims (WNV), Deer tick (DTV) tick-bome encephalitis vims (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever vims (OHFV). In some embodiments, at least some of the non-target protein flavivirus antigens are portions of flavivims non- structural protein 1 (NS1). In some embodiments, at least some of the non-target protein flavivims antigens are portions of flavivims precursor membrane (PrM) protein.

[0127] In certain aspects, the invention provides a high throughput screening method for mapping the peptide that is recognized by a monoclonal antibody within a target protein recognized by the monoclonal antibody, the method including: obtaining a collection of overlapping candidate peptides that are fragments of the protein; providing a high throughput immunoassay device of any one of the claims 35-58; providing at least one immunoassay test strip secured within the high throughput immunoassay device; applying at least one candidate peptide on at least one of the test areas of the at least one test strip, except the uppermost test areas of each of the at least one test strips; applying a control antibody in the uppermost test area of each of the at least one test strip; conjugating a visualizing agent to the monoclonal antibody; applying the visualizing agent conjugated monoclonal antibody in wells of the multi-well plate corresponding the positions of the at least one test strips; immersing the distal end of the at least one test strips into the corresponding wells with the visualizing agent conjugated monoclonal antibody; allowing the visualizing agent conjugated monoclonal antibody to vertically defuse through the test strips and contact the immobilized peptides and the control antibody; incubating the reaction for at least 15-30 minutes; recoding the signal of each test area on the at least one test strip.

[0128] In some embodiments, the control antibody is an antibody that specifically binds the monoclonal antibody. In some embodiments, the control antibody is an antibody against the Fc portion of the monoclonal antibody. In some embodiments, each of the candidate peptides is immobilized on at least one test strip and tested against the visualizing agent conjugated monoclonal antibody.

[0129] In some embodiments, the portion of the test strip that contacts the immobilized candidate peptides is nitrocellulose membrane. In some embodiments, the visualizing reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 600-700 nm. In some embodiments, the species of nanoparticles comprise nanoparticles with an extinction spectral peak at around 500 nm.

[0130] In some embodiments, the target protein is a viral antigen. In some embodiments, the viral antigen is a coronavirus (CoV) antigen. In some embodiments, the coronavirus (CoV) antigen is a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is the SARS-CoV-2 Spike Protein (SARS-CoV- 2S). In some embodiments, the coronavirus (CoV) antigen is the SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0131] In some embodiments, the coronavirus (CoV) antigen is a portion of a protein from a coronavirus (CoV) strain selected from the group consisting of SARS-CoV-2, SARS- CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, the coronavirus (CoV) antigen is a portion of protein from SARS-CoV-2. In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Spike Protein (SARS-CoV-2S). In some embodiments, the coronavirus (CoV) antigen is a portion of SARS-CoV-2 Nucleocapsid Protein (SARS-CoV-2N).

[0132] In some embodiments, the viral antigen is a flavivirus antigen. In some embodiments, the flavivirus antigen is a protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is flavivirus non-structural protein 1 (NS1). In some embodiments, the flavivirus antigen is flavivirus precursor membrane (PrM) protein.

[0133] In some embodiments, the flavivirus antigen is a portion of protein from a flavivirus strain selected from the group consisting of Dengue virus serotype 1 (DV1),

Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). In some embodiments, the flavivirus antigen is a portion of flavivirus non- structural protein 1 (NS1). In some embodiments, the flavivirus antigen is a portion of flavivirus precursor membrane (PrM) protein.

[0134] In some embodiments, the invention provides a method to utilize the high throughput immunoassay device as described above, wherein one group of test strips are utilized to conduct the high throughput screening for antibody paring as described above, and another group of test strips are utilized to conduct the high throughput mapping as described above.

[0135] In some embodiments, the tests utilizing the two groups of test strips are started and interpreted at the same time. In some embodiments, the tests utilizing the two groups of test strips are started or interpreted at different times.

[0136] Lateral Flow Immunoassay

[0137] Lateral flow immunoassays are a cost-effective methodology for detecting a target protein in a biological sample. Tests based on this methodology can be used at a point-of- care center by medical professionals or for at home testing by individuals without any medical or technical training. The technology takes advantage of the capillary properties of materials such as nitrocellulose, which have the capacity to transport fluids spontaneously. Liquids display capillary action, which is their ability to flow through narrow spaces even in opposition to external forces. Capillary action can be observed in the drawing up of liquids into materials such as fibers or cellulose. It occurs due to intermolecular forces between the liquid and surrounding solid surfaces. If the diameter of the space is sufficiently small, then the combination of surface tension of the liquid and the adhesive forces between the liquid and the surrounding solid surface act to propel the liquid into the narrow space.

[0138] There are two main types of lateral flow immunoassays: a “sandwich assay” and a “competitive assay”. The subject matter disclosed herein relates to “sandwich assays.” In a “sandwich assay”, a migrating sample can first come into contact with a detection reagent, which can specifically bind to a target protein or viral antigen. In some embodiments, the detection reagent is conjugated to a visualizing reagent. If the target protein is present in the sample, the conjugated detection reagent can bind to it and subsequently flow laterally a test line (area). The test line (area) may also contain a capture reagent such as proteins or antibodies specific to the target protein. A visual signal or readout is produced when the target protein reaches the test line (area). Thus, the target protein is “sandwiched” between two proteins such as antibodies. [0139] If the detection reagent is not directly labeled and both the capture reagent and the detection reagent are antibodies, then these antibodies must be from different species (i.e., if the capture antibody is a rabbit antibody, the detection antibody can be from goat, chicken, etc., but not rabbit). This will allow the use of a secondary antibody directed to the species heavy chain of the detection antibody. If the detection antibody is directly labeled, then the capture and detection antibodies can be from the same species.

[0140] Usually the main determination of an immunoassay sensitivity is the protein or antibody binding affinity for the target antigen. As the target antigen concentration increases, the amount of detection reagent accumulated in the test line (area) increases, leading to a higher measured response or readout.

[0141] Antibodies

[0142] Monoclonal antibodies (mAb) are generated by identical immune cells that are all clones of a unique parent cell. Monoclonal antibodies can have monovalent affinity - they all bind to the same part of an antigen, called epitope. In contrast, polyclonal antibodies bind to multiple epitopes and are usually made by several different plasma cell lineages. Polyclonal antibodies often contain multiple epitopes and the same affinity purified polyclonal can be used as the capture and labeled detection antibody.

[0143] A paratope is an antigen-binding site found on an antibody which recognizes and binds to an antigen. A paratope usually includes a small region of 5 to 10 amino acids of the antibody's Fc region. The paratope can contain parts of the antibody's heavy and light chains. Each arm of the Y shape of an antibody monomer is tipped with a paratope, which is a set of 6 complementarity-determining regions - 3 of each light chain and heavy chain extending from the fold of antiparallel beta sheets.

[0144] Viral Antieens

[0145] A viral antigen is a protein or a polypeptide encoded by a viral genome. In some embodiments, viral antigens protrude from the virus envelope or capsid and often fulfill important functions, which ensure the survival and propagation of the viral genetic material such as docking to the host cell, fusion, and injection of viral DNA/RNA into a host cell. Recombinant viral antigens can contain part of viral sequence, which encodes a region which can be recognized by antibodies. This reduces the risk of false negatives which can occur with synthetic peptides, which contain only a small portion of the entire protein.

[0146] Coronaviruses (CoVs) are a large family of enveloped, positive-sense, single- stranded RNA viruses that infect a broad range of vertebrates. They are extensive in bats but can be found in many other birds and mammals including humans. In humans, CoVs tend to cause mild to moderate upper respiratory tract infections such as the common cold.

However, in the past couple of decades, there have been outbreaks of severe, and sometimes fatal, respiratory illnesses that were later found to be caused by novel, human pathogenic CoVs. These strains exhibited stronger virulence and quickly passed from human to human. While infection with these CoVs typically produced mild symptoms, for certain individuals, responses were more severe. In extreme cases, death occurred due to gradual respiratory failure as the result of alveolar damage. The seven strain of CoVs found in humans include SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1.

[0147] Virus Families

[0148] Viruses are small parasites that cannot reproduce on their own. Once a virus infects a cell, however, a vims can direct the cell machinery to produce more vimses. Most viruses utilize either RNA or DNA as their genetic material. The nucleic acid may be single- or double-stranded. The infectious vims particle or a virion includes a nucleic acid and an outer shell of protein. The simplest vimses contain only enough RNA or DNA to encode four proteins. On the other hand, the most complex ones can encode up to 200 proteins.

[0149] The nucleic acid of a virion is enclosed within a protein coat called a capsid. The capsid id composed of multiple copies of one protein or a few different proteins, each of which is encoded by a single viral gene. Because of this structure, a vims is able to encode all the information for making a relatively large capsid in a small number of genes.

[0150] Coronavimses (CoVs) are a large family of enveloped, positive-sense, single- stranded RNA vimses that infect a broad range of vertebrates. They are extensive in bats but can be found in many other birds and mammals including humans. In humans, CoVs tend to cause mild to moderate upper respiratory tract infections such as the common cold.

However, in the past couple of decades, there have been outbreaks of severe, and sometimes fatal, respiratory illnesses that were later found to be caused by novel, human pathogenic CoVs. These strains exhibited stronger vimlence and quickly passed from human to human. While infection with these CoVs typically produced mild symptoms, for certain individuals, responses were more severe. In extreme cases, death occurred due to gradual respiratory failure as the result of alveolar damage. The seven strain of CoVs found in humans include SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1.

[0151] The coronavims entry into a cell is mediated by the viral Spike (S) glycoprotein. The Spike protein is a large type I transmembrane protein ranging from 1,160 amino acids to up to 1,400 amino acids. Spike proteins are highly glycosylated with 21 to 35 N- glycosylation sites. Spike proteins assemble into trimers on the virion surface to form the distinctive "corona", or crown-like appearance, of a coronavirus. The ectodomain of all CoV Spike proteins is organized into an N-terminal domain named SI, responsible for receptor binding, and a C-terminal S2 domain, responsible for viral fusion. Coronaviruses can infect the respiratory epithelial cells through interaction with the ACE2 receptor on the cell surface Recombinant Spike protein can bind with recombinant ACE2 proteins.

[0152] The coronavirus Nucleocapsid (N) is a structural protein capable of forming complexes with genomic RNA. Nucleocapsid proteins also interact with the viral membrane protein(s) during virion assembly and play a critical role in enhancing the efficiency of virus transcription and assembly.

[0153] Aedes mosquitoes transmit globally relevant flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV). DENV exists as four antigenic serotypes, DENV1 to DENV4. These viruses have a wide geographic distribution, with approximately 390 million infections annually and more than a quarter of the world’s population at risk.

Prior to 2015, ZIKV was considered obscure and was known to circulate in Africa and Southeast Asia as two separate viral lineages, African and Asian. While most are asymptomatic, the clinical presentation of ZIKV infection resembles that of dengue, including fever, rash, conjunctivitis, arthralgia, and myalgia.

[0154] DENV contains a positive-sense, single-stranded RNA genome of about 10.6 kilobases. The single open reading frame of the DENV encodes a polyprotein precursor, which is cleaved by cellular and viral protease into three structural proteins, the capsid (C), precursor membrane (PrM), and envelope (E), as well as seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. The E protein, a glycoprotein of approximately 55 kDa, contains 12 strictly conserved cysteine residues forming six disulfide bridges and is present as a heterodimer with PrM protein before the maturation of the virion. E protein participates in virus entry and is the major target of both neutralizing and enhancing antibodies.

[0155] The PrM protein is a glycoprotein of about 19 kDa. It contains six highly conserved cysteine residues forming three disulfide bridges. The NS1 protein is also a glycoprotein of about 40 kDa. It contains 12 highly conserved cysteine residues forming six disulfide bridges. NS1 is present intracellularly, on the cell surface, and outside of the cells. [0156] Sequences [0157] SEQ ID NO: 1 is the polypeptide sequence of the coronavirus SARS-CoV-2 Spike protein per gene bank accession NC_045512 and including polymorphisms already reported MT077125 and ancestor QHR63300 RaTG13.

MF VFLVLLPLV S SQC VNLTTRT QLPP AYTN SFTRGVYYPDK VFRS S VLHSTQDLFLPF

FSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQ S

LLI VNNATNVVIK V CEF QF CNDPFLGVY YHKNNK S WME SEFRVY S S ANN CTFEYV S

QPFLMDLEGKQGNFKNLREF VFKNIDGYFKIY SKHTPINL VRDLPQGF S ALEPL VDLP

IGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITD A

VDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEWNATR F

ASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRG

DEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSN

LKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFEL L

HAPATVCGPKKSTNLVKNICCVNFNFNGLTGTGVLTESNfCKFLPFQQFGRDIADTT D

AVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTP T

WRVY ST GSNVF QTRAGCLIGAEHVNNS YECDIPIGAGIC AS YQTQTN SPRRARS VAS

QSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDS TEC

SNLLLQ Y GSFC T QLNRALTGIA VEQDKNT QE VF AQ VKQI YKTPPIKDF GGFNF SQILP

DPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPL LT

DEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLI

AN QFN S AIGKIQD SL S S T AS ALGKLQD VVN QNAQ ALNTLVKQLS SNF GAI S S VLNDIL

SRLDKVEAEVQIDRLITGRLQ SLQT YVTQQLIRAAEIRAS ANL AATKMSEC VLGQ SK

RVDFCGKGYHLMSFPQSAPHGVYFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGV

F VSN GTHWF VT QRNF YEPQIITTDNTF V S GN CD VVIGI VNNT VYDPLQPELD SFKEEL

DKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQY I

KWPWYIWLGFIAGLIAIYMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKG

YKLHYT

[0158] SEQ ID NO: 2 is the polypeptide sequence of the coronavirus Nucleocapsid protein (NCBI Reference Sequence: YP_009724397.2)

MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTAL TQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYY LGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPK GFYAEGSRGGSQAS SRS SSRSRNS SRNSTPGS SRGTSPARMAGNGGDAALALLLLDR LNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQ TQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAD

LDDKDPNFKDQV

ILLNKFQDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQL

QQSMSSADSTQA

[0159] SEQ ID NO: 3 is the polypeptide sequence of the Dengue vims 1 NS1 protein

(NCBI Reference Sequence: NP_722461.1)

DSGCVINWKGRELKCGSGIFVTNEVHTWTEQYKFQADSPKRLSAAIGKAWEEGVCG

IRSATRLENIMWKQISNELNHILLENDMKFTVVVGDVSGILAQGKKMIRPQPMEHKY

SWKSWGKAKIIGADVQNTTFIIDGPNTPECPDNQRAWNIWEVEDYGFGIFTTNIWLK

LRDSYTQVCDHRLMSAAIKDSKAVHADMGYWIESEKNETWKLARASFIEVKTCIWP

KSHTLWSNGVLESEMIIPKIYGGPISQHNYRPGYFTQTAGPWHLGKLELDFDLCEGT T

VVVDEHCGNRGP SLRTTTVT GKTIHEWCCRSCTLPPLRFKGEDGCW Y GMEIRP VKE

KEENLVKSMVSADSGCVINWKGRELKCGSGIFVTNEVHTWTEQYKFQADSPKRLSA

AIGK AWEEGV C GIRS ATRLENIMWKQI SNELNHILLENDMKF TV VV GD V S GIL AQGK

KMIRPQPMEHKYSWKSWGKAKIIGADVQNTTFIIDGPNTPECPDNQRAWNIWEVED

YGFGIFTTNIWLKLRDSYTQVCDHRLMSAAIKDSKAVHADMGYWIESEKNETWKLA

RASFIEVKTCIWPKSHTLWSNGVLESEMIIPKIYGGPISQHNYRPGYFFQFAGPWHL G

KLELDFDLCEGTTVVVDEHCGNRGPSLRTTTVTGKTIHEWCCRSCTLPPLRFKGEDG

C WY GMEIRP VKEKEENLVK SM V S A

[0160] SEQ ID NO: 4 is the polypeptide sequence of the Dengue vims 2 NS1 protein

(NCBI Reference Sequence: NP_739584.2)

DSGCVVSWKNKELKCGSGIFITDNVHTWTEQYKFQPESPSKLASAIQKAHEEGICGI R SVTRLENLMWKQIFPELNHILSENEVKLTIMTGDIKGIMQAGKRSLRPQPFELKYSW KTW GKAKML STESHNQTFLIDGPET AECPNTNRAWN SLEVEDY GF GVFTTNIWLKL KEKQD VF CD SKLM S AAIKDNRA YHADMGYWIES ALND TWKIEK ASFIEVKN CHWP KSHTLWSNGVLESEMIIPKNLAGPVSQHNYRPGYHFQIFGPWHLGKLEMDFDFCDG TTVVVTED CGNRGP SLRTTT AS GKLITEWCCRS C TLPPLRYRGEDGC W Y GMEIRPLK EKEENLVNSLVTA

[0161] SEQ ID NO: 5 is the polypeptide sequence of the Dengue vims 3 NS1 protein

(NCBI Reference Sequence: YP_001531169.2)

DMGCVINWKGKELKCGSGIFVTNEVHTWTEQYfCFQADSPKRLATAIAGAWENGVC GIRSFTRMENLLWKQIANELNYILWENNIKLTVVVGDFLGVLEQGKRFLFPQPMELK Y S WKTWGK AKIVT AET QN S SFIIDGPNTPECP S ASRAWNVWEVED Y GF GVFTTNIWL KLRE VYT QLCDHRLM S AA VKDERA VHADMGYWIES QKN GS WKLEK ASLIEVKTCT WPK SHTL WTN GVLE SDMIIPK SL AGPI S QHNYRPGYHTQT AGP WHLGKLELDFNY C EGTT VVITESCGTRGP SLRTTT V S GKLIHEW C CRS C TLPPLRYMGED GC W Y GMEIRPI SEKEENMVK SL VS A

[0162] SEQ ID NO: 6 is the polypeptide sequence of the Dengue vims 4 NS1 protein (NCBI Reference Sequence: NP_740318.1)

DMGCVASWSGKELKCGSGIFVVDNVHTWTEQYKFQPESPARLASAILNAHKDGVC GIRSTTRLENVMWKQITNELNYVLWEGGHDLTVVAGDVKGVLTKGKRALTPPVSD LKY SWKTW GKAKIFTPEARNSTFLIDGPDTSECPNERRAWN SLEVEDY GFGMFTTNI WMKFREGS SEYCDHRLMSAAIKDQKA VHADMGYWIES SKNQTWQIEKASLIEVKT CL WPKTHTL W SN GVLES QMLIPK S YAGPF S QHNYRQGY ATQT V GP WHLGKLEIDF G ECPGTTVTIQEDCDHRGPSLRTTTASGKLVTQWCCRSCTMPPLRFLGEDGCWYGMEI RPLSEKEENMVKSQVTA

[0163] SEQ ID NO: 7 is the polypeptide sequence of the Asian strain of Zika vims NS1 protein (NCBI Reference Sequence: YP_009430301.1).

DVGC SVDF SKKETRCGT GVF VYND VEAWRDRYKYHPD SPRRLAAAVKQAWEDGIC

GISSVSRMENIMWRSVEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNEL

PHGWKAW GKS YF VRAAKTNN SF VVDGDTLKECPLEHRAWN SFLVEDHGF GVFHTS

VWLKVREDYSLECDPAVIGTAVKGKEAVHSDLGYWIESEKNDTWRLKRAHLIEMK

TCEWPKSHTLWADGIEESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFE E

CPGTK VHVEET C GTRGP SLRSTTAS GRVIEEWCCRECTMPPL SFRAKDGC W Y GMEIR

PRKEPESNLVRSVVTA

[0164] SEQ ID NO: 8 is the polypeptide sequence of the African strain of Zika vims NS1 protein (NCBI Reference Sequence: YP_009227199.1).

DVGC SVDF SKKETRCGTGVFIYND VEAWRDRYKYHPD SPRRL AAAVKQAWEEGIC

GISSVSRMENIMWKSVEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNEL

PHGWKAW GKS YF VRAAKTNN SF VVDGDTLKECPLEHRAWN SFLVEDHGF GVFHTS

VWLKVREDYSLECDPAVIGTAVKGREAAHSDLGYWIESEKNDTWRLKRAHLIEMKT

CEWPK SHTLWTDGVEE SDLIIPK SL AGPL SHHNTREGYRT Q VKGP WHSEELEIRFEEC

PGTKVYVEETCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRP

RKEPESNLVRSMVTA

[0165] SEQ ID NO: 9 is the polypeptide sequence of the Dengue vims 1 PrM protein (NCBI Reference Sequence: NP_733807.2) FHLTTRGGEPHMIV SKQERGK SLLFKT S AGVNMCTLIAMDLGELCEDTMT YKCPRIT ETEPDDVDCWCNATETWVTYGTCSQTGEHRRDKRSVALAPHVGLGLETRTETWMS SEGA WKOTOK VF.TW AT RHPGFTVT AT FT , AHATGTSTTQKGTTFTT 1 MT VTPSM A [0166] SEQ ID NO: TO is the polypeptide sequence of the Dengue vims 2 PrM protein

(NCBI Reference Sequence: NP_739582.2).

FHLTTRNGEPHMIVSRQEKGKSLLFKTEDGVNMCTLMAMDLGELCEDTITYKCPLL RQNEPEDIDC W CNST STWVT Y GTCTTMGEHRREKRS YAL VPHVGMGLETRTETWM SSEGAWKHVQRIETWILRHPGFTMMAAILAYTIGTTHFQRALIFILLTAVTPSMT [0167] SEQ ID NO: 11 is the polypeptide sequence of the Dengue vims 3 PrM protein

(NCBI Reference Sequence: YP_001531166.1)

FHLTSRDGEPRMIVGKNERGKSLLFKTASGINMCTLIAMDLGEMCDDTVTYKCPHIT EVEPEDIDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALAPHVGMGLDTRTQTWMS AEGAWRQVEKVETWALRHPGFTILALFLAHYIGTSLTQKVVIFILLMLVTPSMT [0168] SEQ ID NO: 12 is the polypeptide sequence of the Dengue vims 4 PrM protein

(NCBI Reference Sequence: NP_740315.1)

FSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLGEMCEDTVTYfCCPLL V NFEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSGMGLETRAETWMSS EGAWKHAQRVESWILRNPGFALLAGFMAYMIGQTGIQRTWFVLMMLVAPSYG [0169] SEQ ID NO: 13 is the polypeptide sequence of the Asian strain of the Zika vims

PrM protein (NCBI Reference Sequence: YP_009430297.1)

AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMSYECPM LDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQ TWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYS [0170] SEQ ID NO: 14 is the polypeptide sequence of the African strain of the Zika vims PrM protein (NCBI Reference Sequence: YP_009227197.1)

AEITRRGS A YYMYLDRSD AGKAISF ATTLGVNKCHV QIMDLGHMCDATMS YECPM LDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQ T WLE SRE YTKHLIK VENWIFRNP GF AL V A V AI AWLLGS STS QK VI YL VMILLI AP A Y S

[0171] References

[0172] Bosch I, de Puig H, Hiley M, Carre-Camps M, Perdomo-Celis F, Narvaez CF, Salgado DM, Senthoor D, O'Grady M, Phillips E, Durbin A, Fandos D, Miyazaki H, Yen CW, Gelvez-Ramirez M, Warke RV, Ribeiro LS, Teixeira MM, Almeida RP, Munoz-Medina JE, Ludert JE, Nogueira ML, Colombo TE, Terzian ACB, Bozza PT, Calheiros AS, Vieira YR, Barbosa-Lima G, Vizzoni A, Cerbino-Neto J, Bozza FA, Souza TML, Trugilho MRO, de Filippis AMB, de Sequeira PC, Marques ETA, Magalhaes T, Diaz FJ, Restrepo BN, Marin K, Mattar S, Olson D, Asturias EJ, Lucera M, Singla M, Medigeshi GR, de Bosch N, Tam J, Gomez-Marquez J, Clavet C, Villar L, Hamad- Schifferli K, Gehrke L. 2017. Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum. Sci. Transl. Med. 9, eaanl589.

EXAMPLES

[0173] Example 1 - Approach for developing a rapid diagnostic platform to detect viral antigens

[0174] The stepwise approaches used to develop antigen-based rapid diagnostics are shown in Figure 11 and Table 1. The percentages of amino acid homology and identity among flavivirus NS1 proteins are high (Table 2); therefore, we reasoned that extensive and strategic screening would be required to identify mAbs that detect and distinguish the viruses in a rapid diagnostic test. We tested several commercial anti-dengue NS1 antibodies available from different vendors but found that native NS 1 protein was not recognized or that there was clearly identifiable cross-reactive binding among the DENV serotype NS1 proteins. We therefore generated and characterized anti-NSl antibodies (Figure 11 and Table 1). Groups of mice were injected separately with DENV1-4 recombinant NS1 (rNSl) protein or with ZIKV rNSl protein. B cells from the spleen or lymph nodes were fused with mouse myeloma cells to generate hybridomas. Initial hybridoma screening (209 DENV hybridomas and 104 ZIKV hybridomas) was performed by enzyme-linked immunosorbent assay (ELISA) using individual rNSl protein as antigen (step 1; Table 1). By screening the hybridoma supernatants against individual DENV1-4 NS1 or ZIKV NS1 proteins, the relative ELISA values provided an initial evaluation of differential binding properties for each antibody.

[0175] Table 1. Stepwise description of the approaches used to define antibodies that detect and distinguish DENV1-4 NS1 and the ZIKV NS1 proteins. In Step 3, an 11 X 11 matrix was used to test all combinations of 11 anti-DENV NS1 pairs using each of the four DENVNS1 proteins. In addition, a 10 X 10 matrix was used to test all combinations of 10 anti-ZIKVNSl pairs using ZIKVNS1 protein as well as each of the four DENV serotype NS1 proteins. The matrix-based screening identified optimal monoclonal antibody pairs that detected NS1 without crossover interference. DENV: dengue virus; ZIKV: Zika virus.

AMINO ACID IDENTITY

AMINO ACID HOMOLOGY [0176] Table 2. Amino acid homology and identity among DENV NS1 and ZIKY NS1 proteins. The matrix shows comparisons of percentage amino acid identity (exact positional amino acid matches in the amino acid sequences) and percentage amino acid homology (positional amino acid substitutions by amino acids with similar physicochemical properties) for the DENV and ZIKV NS1 proteins. Dengue virus and Zika virus NS1 sequences were extracted from deposited data (Zika: KX702400.1; DENV1 : GUI 31834.1; DENV2: GQ868641.1; DENV3 : KF955487.1; DENV4: GQ868645.1. These sequences represent viruses that were isolated from the same geographic area in years 2001-2016 for a representative sampling. The BLAST algorithm from the National Center for Biotechnology Information, U.S. National Library of Medicine was used to generate the identity and similarity scores among the NS1 proteins.

[0177] Because of the similarities among the DENV NS1 and ZIKV NS1 proteins (Table 2), each group of mice immunized with a single purified rNSl protein yielded a pool of antibodies, showing both selective binding to a single DENV serotype or ZIKV NS1 and cross-reactive DENV and ZIKV antibodies (Figure 12). ZIKVNS1 hybridomas produced from lymph node tissue represented a higher proportion of clones, showing minimal cross reactivity with dengue NS1 or other flavivirus NS1 proteins, as compared to spleen cell hybridomas (Figure 12). Using the relative ELISA values, 11 DENV mAbs and 10 ZIKV mAbs were selected for further analysis. In step 2 (Figure 11 and Table 1), hybridoma supernatants were used to stain permeabilized Vero cells that had been infected with known ZIKV or DENV viral serotypes. Flow cytometric analysis demonstrated that the mAbs recognized native NS1 protein expressed by virus-infected cells and provided a quantitative analysis of cross-reactive binding when ZIKV antibodies were used to stain DENV-infected cells and vice versa. The 11 DENV mAbs and 10 ZIKV mAbs recognized NS1 protein present in the virus-infected cells; therefore, the hybridomas were expanded, and the mAb isotypes were defined in preparation for affinity chromatography purification.

[0178] The purified antibodies were tested in immunochromatography pairs (step 3; Figure 11 and Table 1), with one antibody conjugated to gold nanoparticles and one antibody adsorbed to nitrocellulose membrane. The 11 DENV hybridomas were tested in a matrix for interactions with DENV NS1 serotypes 1 to 4, ZIKV NS1, or without added antigen as a control. The 10 ZIKV mAbs were also tested in a matrix using ZIKV NS1, a mixture of the four DENV serotype NS1 proteins, or no antigen as a control. We tested 726 DENV combinations (11 x 11 x 6 = 726) and 300 ZIKV combinations (10 x 10 x 3 = 300). Testing throughput was increased by using the half-strip dipstick format, where dipsticks are run in rapid format (about 20 min, depending on humidity conditions) by placing them in microcentrifuge tubes containing small-volume suspensions of conjugated nanoparticles and sample without need for sample paper pads and conjugate paper pads that are characteristic of lateral flow chromatography. The DENV and ZIKV matrices and the immunochromatography results are shown in Figure 13. Eight DENY mAbs and two ZIKV mAbs were ultimately incorporated into the rapid tests used to analyze patient samples. The 10 mAb names, their relative NS1 recognition values in initial screening, and the final application in the rapid tests are shown in Fig. 11 and Table 3.

[0179] Table 3. List of mAbs used in the rapid tests, relative binding values, and summary of final use in the DENV and ZIKV immunochromatography tests. The antibody name is shown at the left column with the antigen (ag) that was used to inject the mice to generate the antibody. The immunochromatography application of each antibody (conjugated to nanoparticle or adsorbed to nitrocellulose membrane) is in the right column. The center columns show “fold over background” (FOB) ELISA values for each of the antigens used as bait on the ELISA plates (DENV 1-4 NS1; upper, or ZIKVNS1 or pooled flavivirus NS1 panel; lower). These numbers define differential recognition of each of the four dengue NS1 antigens during initial screening (Figure 11, step 1). This differential recognition formed the initial selection of the antibodies for use in the final test

[0180] Example 2 - Linear peptide epitope mapping: Mechanisms of specific mAb- NS1 interactions

[0181] To begin to define mechanisms of specific mAb-NSl interactions, we performed linear peptide epitope mapping (step 4; Figure 11 and Table 1). Libraries of tiled DENV NS1 peptides were spotted onto nitrocellulose membranes and incubated with antibody. After washing, positive signals were detected using an anti-mouse IgG antibody coupled to horseradish peroxidase for signal development. In a second approach, tiled peptides were synthesized on glass slides and incubated with each of the antibodies. Positive signals were detected by immunofluorescence microscopy and were scored. The epitope mapping data are summarized in Fig. 14. mAb 7724.323 (“323”) fits the definition of a pan-DENV NS1 antibody because it recognizes all four serotype NS1 proteins in the epitopes 109 to 124 amino acid region. Six antibodies used in the rapid tests (using suffix nomenclature only: 55, 323, 1, 130, 110, and 271) recognize the amino acids 109 to 124 epitope of the “wing” domain (amino acids 30 to 180). However, with the exception of a few pairs with mAb 243, these six antibodies do not recognize the ZIKV NS1 protein (Figure 14 B and 13). The epitope mapping helped to explain the observed antibody specificity in the rapid tests. The shared DENV1-4 NS1 region D/ELKYSWKTWG (amino acids 110 to 119) is not conserved in ZIKVNSl; rather, the comparable ZIKVNS1 region has a distinct amino acid sequence (Figure 14 B), explaining how DENV NS1 mAbs, represented by the members of the 1/55/110/130/271/323 mAb group, do not cross-react with the ZIKV NS1 protein. mAb 243, which showed high specificity for DENV2 (tables S3 and S4), recognized peptides 313 to 330 in DENV2 NS1 (Figure 14 B). The screening approaches, combined with the epitope mapping, contributed to selecting antibodies representing positional epitope diversity; that is, epitopes in both the wing/wing connector/finger and b-ladder domains (Figure 14).

[0182] Matrix-based screening (Figure 13) revealed that the following pairs (nanoparticle/membrane) had optimal rapid test specificity: DENV1, 912/271; DENV2, 243/1; DENV3, 411/55; DENV4, 626/55. In addition, the 912 and 243 antibodies displayed excellent single serotype specificity for the DENV1 and DENV2 proteins, respectively.

These antibodies might be predicted to work as 912/912 and 243/243 homo-pairs in detecting DENV1 and DENV2 NS1 dimers. However, experimental results showed that the limits of NS1 detection were improved by using hetero-pairs without sacrificing specificity; therefore, 912 was paired with 271, and 243 was paired with 1. For ZIKV NS1 detection, we used the 130/110 mAh pair, where both antibodies arose from the lymph node tissue approach (Figure 12). The 411, 55, and 626 antibodies did not display the single serotype specificity observed with 912 and 243 (Table 3); however, when used as pairs in the rapid tests, 411/55 (DENV3) and 626/55 (DENV4) showed excellent specificity. The mAb 323 recognized DENV1-4, however, we found that when used as a 323/323 homo-pair, the detection of DENV4 NS1 in the pan-DENV test was not optimal. Therefore, by conjugating a mixture of mAb on the pan antibody nanoparticles (271, 243, 411, and 626), we achieved improved limit of detection results in the pan-DENV dipstick test. The linear peptide epitope mapping data provide an important framework for understanding the mechanisms of serotype-specific DENV detection and differential detection of DENV and ZIKV.

[0183] Example 3 - Detection of native virus (SARS-CoV-2) Nucleocapsid and spike antigens

[0184] Figure 7 shows a high throughput screen that identifies monoclonal antibody pairing to detect specific viral targets. In the left panel, the test areas are loaded with the immobilized antibodies a-mouse Fc (control antibody), a-SARS-CoV-2 S, and a-SARS- CoV-2 N. The antibodies a-SARS-CoV-2 S and a-SARS-CoV-2 S are also conjugated to nanoparticles. The peptides SARS-CoV-2 S and N are also introduced to the reactions. In the right panel, the test areas are (left to the splitters): a-human Fc (control antibody), a-DENV-2 NS1, and a-SARS-CoV-2 S. The test areas right to the splitters are a-mouse Fc (control antibody), a-DENV-3 NS1, and a-SARS-CoV-2 N. The conjugated antibodies are a-DENV- 2 NS1, a-DENV-3 NS1, a-SARS-CoV-2 S, and a-SARS-CoV-2 N. The antigens introduced to the reactions are DENV-2 NS1, SARS-CoV-2 S, and SARS-CoV-2 N.

[0185] Example 4 - Monoclonal antibody pairs that detect SARS-Coronaviruses [0186] Figure 8 shows a high throughput screen that identifies monoclonal antibody pairs that detect SARS-Coronaviruses with varying levels of specificity and strength.

[0187] Example 5 - Matrix of mAb pair trials for S protein

[0188] Figure 9 shows a high throughput screen to map protein peptides recognized by monoclonal antibodies. Four peptides corresponding to different portions ofDEV S-1 were immobilized on the dipstick test areas. The upper right rest area was loaded with a control antibody a-mouse Fc. Conjugated antibody a-DENV NS 1 was allowed to bind to the immobilized peptides and control antibody.

[0189] Example 6 - Matrix of mAb pair trials for S protein [0190] To define functional antibody pairs, one antibody was conjugated to gold nanoparticles, and one antibody was adsorbed to nitrocellulose membrane. The resulting nanoparticle conjugates-membrane pairs were tested using the specific serotype recombinant S protein and S protein domains (S protein, Receptor-binding Domain of the S protein (RBD), N-terminal domain of the S protein (NTD)).

[0191] Figure 10 shows an antibody grid from a high throughput screening.

[0192] Table 4. Materials