Development of a filovirus immunochromatographic lateral flow assay and assay device

Abstract

A rapid antigen test capable of detecting Ebola virus, Sudan virus, Bundibugyo virus, Tai Forest virus, Bombali virus and Reston virus was generated. The test relies on two or more monoclonal antibodies that are specific for the glycoprotein of these viruses that can be paired together, that is, monoclonal antibodies that recognize different regions of the glycoprotein whose binding does not interfere with each other. In one example, monoclonal antibody 6H8 is conjugated to colloidal gold and serves as the detection antibody, while monoclonal antibody 5A3 is sprayed on the test line and serves as the capture antibody.

Description

[0001] DEVELOPMENT OF A FILOVIRUS IMMUNOCHROMATOGRAPHIC LATERAL FLOW ASSAY AND ASSAY DEVICE PRIOR APPLICATION INFORMATION

[0002] The instant application claims the benefit of US Provisional Patent Application 63 / 672,450, entitled “DEVELOPMENT OF A FILOVIRUS IMMUNOCHROMATOGRAPHIC LATERAL FLOW ASSAY”, and filed July 17, 2024, the entire contents of which are hereby incorporated herein by reference for all purposes.

[0003] BACKGROUND OF THE INVENTION

[0004] Accurate, efficient, and rapid diagnostic tools are essential to address outbreaks of high consequence zoonotic pathogens, both in Canada and around the world. Currently, the most widely used method of rapid diagnosis is quantitative polymerase chain reaction (qPCR); however, this assay suffers from the requirements for (1) specially trained personnel for sample processing; (2) expensive equipment, reagents, and disposables; (3) dedicated laboratory space for running the test; and (4) longer wait-times for results. There is, therefore, a need to develop rapid, simple, and inexpensive diagnostic tests that can be deployed at the point-of-care in potentially resource-poor locations that are capable of detecting orthoebolavirus antigen in easily acquired clinical samples (such as saliva, blood, or serum).

[0005] SUMMARY OF THE INVENTION

[0006] According to an aspect of the invention, there is provided an immunochromatographic lateral flow assay device for detecting Ebola virus glycoprotein, Sudan virus glycoprotein, Bundibugyo virus glycoprotein, Tai Forest virus glycoprotein, Bombali virus glycoprotein and / or Reston virus glycoprotein in a sample comprising:

[0007] (a) a sample pad for receiving the sample to be tested;

[0008] (b) a conjugate release pad pre-treated with a suitable buffer, said conjugate release pad comprising at least one detection antibody for detecting an orthoebolavirus antigen within the sample;

[0009] (c) a nitrocellulose pad comprising at least one paired capture antibody; and

[0010] (d) an absorption pad for absorbing the sample, said device configured such that once a sample is applied, the sample flows in order from (a) to (b) to (c) to (d), wherein the detection antibody and the paired capture antibody is selected from the group consisting of: a monoclonal capture antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:46 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:29 (5A3) and a monoclonal detection antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:57 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NQ:40 (6H8); a monoclonal capture antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:58 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:41 (6F12) and a monoclonal detection antibody 6H8; a monoclonal capture antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:53 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:36 (1H11) and a monoclonal detection antibody 6H8; a monoclonal capture antibody 6H8 and a monoclonal detection antibody produced by cell line 6F12; a monoclonal capture antibody 6H8 and a monoclonal detection antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:59 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:42 (4F9a) or to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NQ:60 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:43 (4F9b); a monoclonal capture antibody 6H8 and a monoclonal detection antibody produced by cell line 1 H11 ; and a monoclonal capture antibody 6H8 and a monoclonal detection antibody 5A3.

[0011] According to an aspect of the invention, there is provided an immunochromatographic lateral flow assay device for detecting Ebola virus glycoprotein, Sudan virus glycoprotein, Bundibugyo virus glycoprotein, Tai Forest virus glycoprotein, Bombali virus glycoprotein and / or Reston virus glycoprotein in a sample comprising:

[0012] (a) a sample pad for receiving the sample to be tested; (b) a conjugate release pad pre-treated with a suitable buffer, said conjugate release pad comprising at least one detection antibody selected from the group consisting of: a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:49 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:32 (1A3); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NQ:50 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:33 (1 E3); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:52 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 35 (1H5); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:47 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NQ:30 (4A2); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:51 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:34 (4H6); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:58 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:41 (6F12); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:48 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:31 (6H1); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:46 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:29 (5A3); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO: 54 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:37 (1 D12); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:61 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:44 (1G1); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:59 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:42 (4F9a) or to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NQ:60 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:43 (4F9b); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:62 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:45 (1G3); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:53 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:36 (1 H11); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:57 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NQ:40 (6H8); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:56 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 39 (1A9); and a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:55 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:38 (3F7); for detecting an orthoebolavirus antigen within the sample;

[0013] (c) a nitrocellulose pad comprising at least one paired capture antibody selected from the group consisting of 1A3; 1 E3; 1H5; 4A2; 4H6; 6F12; 6H1 ; 5A3; 1 D12 ; 1G1 ; 4F9; 1G3; 1H11 ; 6H8; 1A9; and 3F7; said at least one paired capture antibody being different from the at least one detection antibody but binding to the orthoebolavirus antigen independently and without interfering with the binding of the at least one detection antibody to the antigen

[0014] (d) an absorption pad for absorbing the sample, said device configured such that once a sample is applied, the sample flows in order from (a) to (b) to (c) to (d).

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1. Lateral Flow Test Strip components and assembly (Image source: https: / / lateralflows.com / lateral-flow-assays / ). In general, lateral flow test strips are made up of several components. Upon a backing card (used for structural support), a nitrocellulose membrane is applied, flanked by a sample and conjugate pad on one side and an absorption pad on the other. The Capture monoclonal antibody (mAb) is applied in a thin line to the nitrocellulose membrane to form the Test line, while a control mAb is applied in a thin line to form the Control line. The gold-conjugated Detection mAb is applied to the conjugate pad. Liquid sample is applied directly to the sample pad, and, through capillary action, flows across the conjugate pad and nitrocellulose membrane towards the absorption pad. Detection mAb in the conjugate pad binds to antigen in the sample as it flows. The same antigen then binds to the Capture mAb at the test line, concentrating the antigen-Detection mAb complex and producing a visual signal — typically a dark red or grey-coloured line formed by the gold conjugated to the Detection mAb. Detection mAb that is not bound to antigen continues flowing towards the control line, which contains antibody specific for the Detection mAb. Appearance of a visual line here confirms that the test has operated correctly.

[0017] Figure 2. Bio-layer interferometry (BLI) using the Sartorius Octet instrument identifies potential capture and detection mAbs for a pan-orthoebolavirus ILF test. Streptavidin (SA) sensors loaded with biotinylated monoclonal antibodies (mAbs), mirroring gold-conjugated detection mAbs, were exposed to 500 nM recombinant Ebola virus (EBOV, IBT 0501-015), Sudan virus (SUDV, IBT 0502-025) or Bundibugyo virus (BDBV, IBT 0505- 015) glycoprotein (GP), followed by exposure to a second, unconjugated mAb, mirroring the capture mAb. The association and dissociation of the second mAb was quantified.

[0018] Figure 3. Bio-layer interferometry (BLI) identifies 5A3 as capture mAb and 6H8 as detection mAb for the pan-orthoebolavirus ILF test. A Sartorius Octet instrument was used to quantify the sequential binding of antibodies to recombinant Ebola virus (EBOV, IBT 0501-015), Sudan virus (SUDV, IBT 0502-025), and Bundibugyo virus (BDBV, IBT 0505-015) glycoprotein (GP). Streptavidin (SA) sensors loaded with biotinylated monoclonal antibodies (mAbs), mirroring gold-conjugated detection mAbs, were exposed to 500 nM recombinant GP, followed by exposure to a second, unconjugated mAb, mirroring the capture mAb. The association and dissociation of the second mAb was quantified.

[0019] Figure 4. mAbs 5A3 and 6H8 form a suitable pair for a pan-orthoebolavirus ILF.

[0020] Six capture-detection mAb pairs were evaluated in an unoptimized ILF test using 50 L of Ebola virus / Makona C07 (EBOV; infectious titer of 36,350 median tissue culture infectious dose (TCID50)), Sudan virus / Gulu (SUDV; 6,000 TCID50), Bundibugyo virus / Uganda (BDBV; 5,000 TCID50), Reston virus (RESTV; 2,505 TCID50) or Marburg virus / Angola 325 (MARV; 15,000 TCID50) combined with 50 pL PBS. The test and control lines are indicated with T and C, respectively.

[0021] Figure 5. The 5A3-6H8 pan-orthoebolavirus ILF test detects low concentrations of recombinant glycoproteins. Recombinant glycoprotein (GP) or soluble glycoprotein (sGP) from Ebola virus (EBOV, IBT 0501-001 , IBT 0565-001 , respectively), Sudan virus (SUDV, IBT 0502-001 , IBT 0570-001 , respectively), Bundibugyo virus (BDBV, IBT 0505-015), or Marburg virus (MARV, IBT 0506-015) was serially diluted from 500 to 31.2 ng in sample running buffer and spiked with 10 pL per test strip of plasma or whole blood from a human donor (CE0010253; see Figure 6). A total of 100 pL was applied to the ILF test. The test and control lines are indicated with T and C, respectively.

[0022] Figure 6. Commercially obtained plasma and whole blood from human donors. Plasma and whole blood from six diverse individuals were obtained from StemCell. All samples gave negative results on the 5A3-6H8 pan-orthoebolavirus ILF test, and a subset of the samples were spiked with recombinant glycoprotein (GP) or virus to further assess ILF performance. The plasma and blood samples that are diversified in age, gender, ethnicity and blood type indicates that this ILF test is highly compatible with human blood samples.

[0023] Figure 7. The 5A3-6H8 pan-orthoebolavirus ILF test detects authentic EBOV, SUDV, BDBV, and RESTV in different carriers. Ebola virus / Makona C07 (EBOV; infectious titer of 36,350 media tissue culture infections dose (TCID50)), Sudan virus / Gulu (SUDV; 6,000 TCID50), Bundibugyo virus / Uganda (BDBV; 5,000 TCID50), Reston virus (RESTV; 2,505 TCID50), Marburg virus / Angola 325 (MARV; 15,000 TCID50), Lassa virus (LASV; 50,000 TCID50), or SARS-CoV-2 (5,000 TCID50) were added to sample running buffer and spiked with 10 pL of plasma or whole blood (donor CE0010253; see Figure 6) per test strip. A total of 100 pL was applied to the 5A3-6H8 pan-orthoebolavirus ILF test. The test and control lines are indicated with T and C, respectively.

[0024] Figure 8. The 5A3-6H8 pan-orthoebolavirus ILF test detects low concentrations of infectious EBOV, SUDV, BDBV, and TAFV. Ebola virus / Makona C07 (EBOV), Sudan virus / Gulu (SUDV), Bundibugyo virus / Uganda (BDBV), Reston virus (RESTV), and Tai Forest virus (TAFV) were serially diluted two-fold in sample running buffer from a starting concentration of 6000 median tissue culture dose (TCID50) or 34,620 TCID50 (EBOV only), and 100 pL of each dilution was applied to each test strip. The test and control lines are indicated with T and C, respectively. The cycle threshold (Ct) values from RT-qPCR assays targeting the L gene, or E gene for SARS-CoV-2, in total RNA extracted from whole blood samples are provided for comparison.

[0025] Figure 9. The 5A3-6H8 pan-orthoebolavirus ILF test detects virus in blood samples from experimental animals. Whole blood samples from ferrets infected with Ebola virus / Makona C07 (EBOV), Sudan virus / Gulu (SUDV), Bundibugyo virus / Uganda (BDBV), or recombinant EBOV expressing Bombali virus (BOMV) GP instead of EBOV GP (EBOV- BOMV GP) and nonhuman primates (NHPs) infected with EBOV / Makona C07 were tested at various timepoints (i.e. , days post infection, dpi) during the course of the infection, as indicated. A total of 10 pL whole blood was applied to the test strip and run with 100 pL of sample running buffer. The urea concentration of the sample running buffer was doubled for NHPs to avoid false positive results. The cycle threshold (Ct) values from RT-qPCR assays targeting the L gene in total RNA extracted from whole blood samples are provided for comparison. ND, not detected.

[0026] Figure 10. The 5A3-6H8 pan-orthoebolavirus ILF test does not detect EBOV variant Makona C05. (A) Ebola virus / Mayinga (EBOV; infectious dose of 150,000 median tissue culture infectious dose (TCID50)), EBOV / Kikwit (64,700 TCID50), EBOV / Makona C07 (72,700 TCID50), and EBOV / Makona C05 (60,000 TCID50) were diluted 1:1 with 2X sample running buffer, and 100 pL were applied to the ILF test. (B) A segment of the glycoprotein amino acid sequence alignment is shown for a representative subset of orthoebolaviruses, highlighting the unique polymorphism W291T in EBOV / Makona C05 that presumably prevents detection by the ILF test. (C) This W291R polymorphism is only present in EBOV / Makona C05 variant, which accounts for a total of 14 (0.5%) orthoebolavirus sequences deposited in the NCBI data base.

[0027] Figure 11. The 5A3-6H8 pan-orthoebolavirus ILF test detects SUDV variants Boneface and Gulu, but does not detect MARV variants Angola and Musoke or RAW. (A) Sudan virus (SUDV) variants Boneface (infectious titer of 6,000 median tissue culture infectious dose (TCID50)) and Gulu (6,000 TCID50) were diluted 1:1 with 2X sample running buffer, and 100 pL were applied to the ILF test. (B) Marburg virus (MARV) variants Angola 325 (158,000 TCID50) and Musoke (28,100 TCID50), as well as Ravn virus (RAW; 232,200 TCID50) were diluted 1 :1 with 2X sample running buffer, and 100 pL were applied to the ILF test.

[0028] DESCRIPTION OF THE PREFERRED EMBODIMENTS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

[0029] Described herein is a rapid antigen test capable of detecting Ebola virus, Sudan virus, Bundibugyo virus, TaT Forest virus, Bombali virus and Reston virus.

[0030] In some embodiments, as discussed herein, the test comprises at least one monoclonal detection antibody selected from the group consisting of: 1A3; 1E3; 1H5; 4A2; 4H6; 6F12; 6H1; 5A3; 1D12 ; 1G1; 4F9; 1G3; 1H11; 6H8; 1A9; and 3F7; for detecting an orthoebolavirus antigen within the biological sample; and at least one paired monoclonal capture antibody selected from the group consisting of 1A3; 1E3; 1H5; 4A2; 4H6; 6F12; 6H1; 5A3; 1D12 ; 1G1; 4F9; 1G3; 1H11; 6H8; 1A9; and 3F7; said at least one paired capture antibody being different from the at least one detection antibody but binding to the orthoebolavirus antigen independently and without interfering with the binding of the at least one detection antibody to the orthoebolavirus antigen.

[0031] In some embodiments, as discussed herein, the test comprises at least one monoclonal detection antibody selected from the group consisting of: 1H5; 4H6; 6F12; 6H1 ; 5A3; 1D12 ; 1G1; 4F9; 1G3; 1H11; 6H8; 1A9; and 3F7; for detecting an orthoebolavirus antigen within the biological sample; and at least one paired monoclonal capture antibody selected from the group consisting of 1H5; 4H6; 6F12; 6H1 ; 5A3; 1D12 ; 1G1 ; 4F9; 1G3; 1 H11 ; 6H8; 1 A9; and 3F7; said at least one paired capture antibody being different from the at least one detection antibody but binding to the orthoebolavirus antigen independently and without interfering with the binding of the at least one detection antibody to the orthoebolavirus antigen.

[0032] The nucleotide sequences of the heavy chain variable regions of select monoclonal antibodies are provided below: 5A3_Heavy Chain_nt CAGGTCCAGCTGCAGCAGTCTGGGCCTGAGCTGGTGAGGCCTGGGGTCTCAGTGAAGA TTTCCTGCAAGGTTTCCGGCTACACATTCACTGATTATGCTATGCACTGGGTGAAGCAGA GTCATGCAAAGAGTCTAGAGTGGATTGGAGTTATTAGTACTTACTCTGGTAATACAAAGT ACAACCAGAACTTTAAGGGCAAGGCCACAATGACTGTAGACAAATCCTCCAGCACAGCC TATATGGAACTTGCCAGATTGACATCTGAGGATTCTGCCATCTATTACTGTGCGCTGGGG GGCCCCCGGGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG (SEQ ID NO:1)

[0033] F3-4A2-5-HC - 4A2 Heavy chain

[0034] CAGGTCCAGCTGCAGCAGTCTGGACCTGAACTGGTAAGGCCTGGGACTTCAGTGAAGG

[0035] TTTCCTGCAAGGCTTCTGGATACGCCTTCACTAATTATTTGATAGAGTGGCTAAGGCAGA GGCCTGGACAGGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGTGGTGGTACTAAT TACAATGAGAAATTCAAGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAGCACTGC

[0036] CTACATGCGGCTCGGCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGTGCAAGAG CACTACGTGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC (SEQ ID NO:2)

[0037] F3-6H1-6-HC - 6H1 Heavy chain

[0038] CAGGTCCAGCTGCAGCAGTCTGGACCTGAACTGGTAAGGCCTGGGACTTCAGTGAAGG

[0039] TTTCCTGCAAGGCTTCTGGATACGCCTTCACTAATTATTTGATAGAGTGGCTAAGGCAGA GGCCTGGACAGGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGTGGTGGTACTAAT TACAATGAGAAATTCAAGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAGCACTGC

[0040] CTACATGCGGCTCGGCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGTGCAAGAG CACTACGTGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC (SEQ ID NO:3)

[0041] F3-1A3-2-HC - 1A3 Heavy chain

[0042] CAGGTCCAGCTGCAGCAGTCTGGACCTGAACTGGTAAGGCCTGGGACTTCAGTGAAGG

[0043] TTTCCTGCAAGGCTTCTGGATACGCCTTCACTAATTATTTGATAGAGTGGCTAAGGCAGA GGCCTGGACAGGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGTGGTGGTACTAAT TACAATGAGAAATTCAAGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAGCACTGC

[0044] CTACATGCGGCTCGGCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGTGCAAGAG CACTACGTGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC (SEQ ID NO:4)

[0045] F3-1E3-4-HC - 1E3 Heavy chain

[0046] CAGGTCCAGCTGCAGCAGTCTGGACCTGAACTGGTAAGGCCTGGGACTTCAGTGAAGG

[0047] TTTCCTGCAAGGCTTCTGGATACGCCTTCACTAATTATTTGATAGAGTGGCTAAGGCAGA GGCCTGGACAGGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGTGGTGGTACTAAT TACAATGAGAAATTCAAGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAGCACTGC

[0048] CTACATGCGGCTCGGCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGTGCAAGAG CACTACGTGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC (SEQ ID NO:5)

[0049] F3-4H6-2-HC - 4H6 heavy chain

[0050] CAGATCCAGTTGGTGCAGTCTGGACCTGAACTGAAGAAGCCTGGAGAGACAGTCAAGAT CTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTATTCAATTCACTGGGTGAAGCAGGC TCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCAACAT ATGCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCT ATGTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCCATAGACC ATTCCTTCTATGGTGACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCT CC (SEQ ID NO:6)

[0051] F3-1H5-4-HC_consensus sequence - 1 H5 heavy chain GAGGTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGTCTTCCGTGAAGA TGTCCTGCAAGGCTTCTGGCTACAGCTTAACCTACTATTGGATGCACTGGGTAAGACAGA GGCCTGGACAGGGTCTAGAATGGATTGGTTCTATTTATCCTAGAAATAGTGATACTAACT ACAACCAGAAGTTCAAGGGCAAGGCCAAACTGACTGCAGTCACATCCGCCAACACTGCC TACATGGAACTCAGCAACCTGACAAATGAGGACTCTGCGGTCTATTACTGTTCAAGAGAG CCTTACTATAAGTACGACCGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGT

[0052] CTCC (SEQ ID NO:7)

[0053] F6-1H11-7-HC - 1H11 heavy chain

[0054] GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGAT

[0055] ATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGA GCCATGGAAAGAACCTTGAGTGGATTGGACTTATTAATCCTTACAATGGTGGTACTACCT ACAACCAGAAGTTCAAGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAGCACAGCC

[0056] TACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGT GGATCTACTATGATTACGACGACGATGACGGGGCTATATTACTTTGCTATGGACTACTGG GGTCAAGGAACCTCAGTCACCGTCTCC (SEQ ID NO:8)

[0057] F5-1D12-1-HC - 1D12 heavy chain

[0058] GAGGTTCAGCTGCAGCAGTCTGGGGCAGAACTTGTGAGGCCAGGGGCCTCAGTCAAAT

[0059] TGTCCTGCACAGCTTCTGGCTTCAACATTGAAGACACCTATATACATTGGGTGAAGCAGA GGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTGAA TATGACCCGAAGTTCCAGGGCAAGGCCACTATAACTTCAGACACATCCTCCAACACAGT CTTCCTGCAGCTCAGCAACCTGACATCTGAGGACACTGCCGTCTACTTCTGTGCCCAGG

[0060] GTGGACTCTGGGGCCACGGCACCGTTCTCACAGTCTCC (SEQ ID NO:9)

[0061] F5-3F7-11-HC - 3F7 heavy chain

[0062] GAGGTTCAGCTGCAGCAGTCTGGGGCGGAGCTTGTGAAGCCAGGGGCCTCAGTCAGGT TGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATATACACTGGGTGAAGCAGA GGCCTCAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATAAAAAA TATGACCCGAAGTTCCAGGGCAAGGCCACTATAACATCAGACACATCCTCCAACACAGC CTACCTCCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCCCAGG

[0063] GTGGACTCTGGGGCCAAGGCACCGCTCTCACAGTCTCC (SEQ ID NO: 10)

[0064] F14-1A9-11-HC - 1A9 heavy chain

[0065] CAGGTTCAGCTCCAGCAGTCTGGGGCTGAGCTGGCAAGACCTGGGGCTTCAGTGAAGT

[0066] TGTCCTGCAAGGCTTCTGGCTACACCTTTACTACCTACTGGCTGCAGTGGGTAAAACAGA GGCCTGGACAGGGTCTGGAATGGATTGGGTCTATTTATCCTGGAGATGGTGATACAAAA TATATTCAGAAGTTCAAGGACAAGGCCACAATGACTGCAGATACCTCCTCCAGCACAACC

[0067] TACATGCAGCTCAGCAGGTTGGCATCTGAGGACTCTGCGGTCTATTATTGTGCAAGATTG

[0068] ACGTTCGACGGAGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC (SEQ ID NO:11)

[0069] F13-6H8-13-HC - 6H8 heavy chain

[0070] GATGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAAC TCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGTACTGGGTTCGTCAGG CTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAATAGTGGCAGTAGTACCATCTAC

[0071] TATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATACCAAGAACACCCT GTTCCTGCAAATGACCAGTCTGAGGTCTGAAGATACGGCCATATATTACTGTGCAAGAAG GGGCCTTACTCCGGTCTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACC

[0072] GTCTCC (SEQ ID NO: 12)

[0073] F3-6F12-12-HC-consensus sequence - 6F12 heavy chain

[0074] GATGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTACAGCCTGGAGGGTCCCGGAAAC

[0075] TCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGTACTGGGTTCGTCAGG

[0076] CTCCAGAGAAGGGACTGGAGTGGGTCGCATACATTAGTTATGACAGTAATACCATCTACT

[0077] ATGCAGACACAGTGAAGGACCGATTCACCATCTCCAGAGACAATCCCAAGAGCACCCTG

[0078] TTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGATC

[0079] GGGCATTACGACGGTATATTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACC

[0080] GTCTCC (SEQ ID NO: 13)

[0081] F5-4F9-20-HC - 4F9 heavy chain

[0082] GATGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCT

[0083] CACCTGCACTGTCACTGGCTTCTCAATCACCAGTGATTATGCCTGGAACTGGATCCGGC

[0084] AATTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAGCTACAGTGGTATCACTAAAT

[0085] ACAACCCATCTCTCAGAAGTCGAATCTCTATTACTCGAGACACATCCAAGAACCAGTTCT

[0086] TCCTGCAGTTGAATTCTGTGACTCCTGAGGACACAGCCACATATTACTGTGCAAGATTCG

[0087] GTTACGGGGGGGTTTGGGGCCAAGGGACTCTGGTCACTGTCTCT (SEQ ID NO: 14)

[0088] The nucleotide sequences of the light chain variable regions of select monoclonal antibodies are provided below:

[0089] 5A3_Light Chain_nt

[0090] CAAATTGTTCTCACCCAGTCTCCAGCACTCATGTCTGCATCTCCAGGGGAAAAGGTCACC

[0091] ATGACCTGCAGTGCCAACTCAAGTGTAAGTTACATGTACTGGTACCAGCATAAGCCAAGA

[0092] TCCTCCCCCAAAGTCTGGATTTATCTCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGC

[0093] TTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGA

[0094] AGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCACTCACGTTCGGTGCTG

[0095] GGACCAAGCTGGAGCTGAAAC (SEQ ID NO: 15)

[0096] F3-1A3-2-LC - 1A3 light chain

[0097] GATGTTGTTCTGACCCAAACTCCACTCTCTCTGCCTGTCAATATTGGAGATCAAGCCTCT

[0098] ATCTCTTGCAAGTCTCCTAAGAGTCTTCTAAATAGTGATGGATTCACTTATTTGGACTGGT

[0099] ACCTGCAGAAGCCAGGCCAGTCTCCACAGCTCCTAATATATTTGGTTTCTAATCGATTTT

[0100] CTGGAGTTCCAGGCAGGTTCACTGGCAGTGGCTCAGGAACAGATTTCACACTCAAGATC

[0101] AGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTTCCAGAGTAACTATCTTCCA

[0102] TTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCT (SEQ ID NO: 16)

[0103] F3-1E3-4-LC - 1E3 light chain

[0104] GATGTTGTTCTGACCCAAACTCCACTCTCTCTGCCTGTCAATATTGGAGATCAAGCCTCT

[0105] ATCTCTTGCAAGTCTCCTAAGAGTCTTCTAAATAGTGATGGATTCACTTATTTGGACTGGT

[0106] ACCTGCAGAAGCCAGGCCAGTCTCCACAGCTCCTAATATATTTGGTTTCTAATCGATTTT

[0107] CTGGAGTTCCAGGCAGGTTCACTGGCAGTGGCTCAGGAACAGATTTCACACTCAAGATC

[0108] AGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTTCCAGAGTAACTATCTTCCA

[0109] TTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCT (SEQ ID NO: 17)

[0110] F3-1H5-4-LC - 1H5 light chain

[0111] GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAG CATCACCTGCAAGGCCAGTCAGGATGTAAGTACTGCTGTAGCCTGGTATCAACAAAAAC

[0112] CAGGGCAATCTCCTAAACTACTGTTTTACTGGGCATCCACCCGGCACACTGGAGTCCCT

[0113] GATCGCTTCACAGGCAGTGGATCTGGGACAGATTATACTCTCACCATCAGCAGTGTGCA

[0114] GGCTGAAGACCTGGCACTTTATTACTGTCAGCAACACGATAACACTCCATTCACGTTCGG

[0115] CTCGGGGACAAAGTTGGAAATAAAACGGGCT (SEQ ID NO:18)

[0116] F3-4A2-5-LC - 4A2 light chain

[0117] GATGTTGTTCTGACCCAAACTCCACTCTCTCTGCCTGTCAATATTGGAGATCAAGCCTCT

[0118] ATCTCTTGCAAGTCTCCTAAGAGTCTTCTAAATAGTGATGGATTCACTTATTTGGACTGGT

[0119] ACCTGCAGAAGCCAGGCCAGTCTCCACAGCTCCTAATATATTTGGTTTCTAATCGATTTT

[0120] CTGGAGTTCCAGGCAGGTTCACTGGCAGTGGCTCAGGAACAGATTTCACACTCAAGATC

[0121] AGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTTCCAGAGTAACTATCTTCCA

[0122] TTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCT (SEQ ID NO: 19)

[0123] F3-4H6-2-LC - 4H6 light chain

[0124] GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCC

[0125] ATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGT

[0126] ACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTT

[0127] CTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATC

[0128] AGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCC

[0129] GTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCT (SEQ ID NQ:20)

[0130] F3-6F12-12-LC - 6F12 light chain

[0131] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTATCTGAGGGAGAAACTGTCAC

[0132] CATCACATGTCGAGCAAGCGAGAATATTTACAGTAATTTAGCATGGTATCAGCAGAGACA

[0133] GGGAAAATCTCCTCACCTCCTGGTCTATGCTGGAACAAAGTTAGCAGATGGTGTGCCAT

[0134] CAAGGTTCAGTGGCAGTGGATCAGGCACACAGTATTCCCTCAAGATCAACAGCCTGCAG

[0135] TCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGGATACTCCGTGGACGTTCGGT

[0136] GGAGGCACCAAGCTGGAAATCAAACGGGCT (SEQ ID NO:21)

[0137] F3-6H1-6-LC - 6H1 light chain

[0138] GATGTTGTTCTGACCCAAACTCCACTCTCTCTGCCTGTCAATATTGGAGATCAAGCCTCT

[0139] ATCTCTTGCAAGTCTCCTAAGAGTCTTCTAAATAGTGATGGATTCACTTATTTGGACTGGT

[0140] ACCTGCAGAAGCCAGGCCAGTCTCCACAGCTCCTAATATATTTGGTTTCTAATCGATTTT

[0141] CTGGAGTTCCAGGCAGGTTCACTGGCAGTGGCTCAGGAACAGATTTCACACTCAAGATC

[0142] AGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTTCCAGAGTAACTATCTTCCA

[0143] TTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCT (SEQ ID NO:22)

[0144] F5-1D12-1-LC - 1 D12 light chain

[0145] GACATTGTGATGATCCAGTCTCAAAAATTCATGTCCACGTCAATAGGAGACAGGGTCAGC

[0146] GTCACCTGCAAGGCCAGTCAGAATGTGGGTTCTAATGTAGCCTGGTATCAACAGAAATC

[0147] AGGGCAATCTCCTAAAGCAGTGATTTATTCGGCATCCAACCGGTACAGTGGAGTCCCAG

[0148] ATCGCTTCACAGGCCGTGGATCTGGGACAGATTTCACTCTCACCATCACCAATGTGCAG

[0149] TCTGAAGACTTGGCAGAGTATTTCTGTCAACAATATAGTACCTATCCATTCACGTTCGGCT

[0150] CGGGGACAAAGTTGGAAATAAAACGGGCT (SEQ ID NO:23)

[0151] F5-3F7-11-LC - 3F7 light chain

[0152] GACATTGTGATGATGCAGTCTCAGAAATTCATGTCCACATCAGTAGGAGACAGGGTCAG

[0153] CGTCACCTGCAAGGCCAGTCAGAATGTGGGTAGTAAAGTAGCCTGGTATCAACAGAAAC CAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTCCCGGTACAGTGGAGTCCCT GATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCTATGTGCA GTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCATTCACGTTCGG

[0154] CTCGGGGACAAAGTTGGAAAGAAAACGGGCT (SEQ ID NO:24)

[0155] F5-4F9-20-LC - 4F9 light chain

[0156] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTGACCATTGGACAACCAGCCTC

[0157] CATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAGGACATATTTGAATTG

[0158] GTTTTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGA TTTTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAAT CAGCAGAGTGGAGGCTGAGGATTTGGGAATTTATTATTGTTGGCAAGGTACACATTTTCC

[0159] TCAGACGTTCGGTGGAGGCTCCAAGCTAGAAATCAAACGGGCT (SEQ ID NO:25)

[0160] F6-1H11-7-LC - 1H11 light chain

[0161] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCAC

[0162] CATCACATGTCGAGCAAGTGGGAATATTCACAATTCTTTAGCATGGTATCAGCAGAAACA

[0163] GGGAAAATCTCCTCGGCTCCTGGTCTATTATGCAAAGACCTTAGCAGATGGTGTGCCAT

[0164] CAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAG CCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGAGTACTCCCTACACGTTCGGA

[0165] GGGGGGACCAAGCTGGAAATAAAACGGGCT (SEQ ID NO:26)

[0166] F13-6H8-13-LC - 6H8 light chain

[0167] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTATCTGTGGGAGAAACTGTCACC

[0168] ATCACATGTCGAGCAAGTGAGAATATTTACAATAATTTAGCATGGTTTCAGCAGAAACAG

[0169] GGAAAATCTCCTCAGCTCCTAGTCTATGCTGCAACAAAATTAGCAGATGGTGTGCCATCA AGGTTCAGTGGCAGTGGATCAGGCACACAGTATTCCCTCAAGATCAACAGCCTGCAGTC

[0170] TGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGGATATTCCGTGGACGTTCGGTGG

[0171] AGGCACCAAGCTGGAAATCAAACGGGCT (SEQ ID NO:27)

[0172] F14-1A9-11-LC - 1A9 light chain

[0173] GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCAC

[0174] TATGAGCTGCAAATCCAGTCAGAGTCTGCTCAGCAGTAGAACCCGAAAGAACTACTTGG

[0175] CTTGGTATCAGCAGAAACCAGGGCAGTCTCCTAAATTACTGATCTACTGGGCATCCACTA GGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTC ACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAAT

[0176] TTCACGTTCGGCTCGGGGACAAAGTTGGAGATAAAACGGGCT (SEQ ID NO:28)

[0177] The amino acid sequences of the heavy chain variable regions of select monoclonal antibodies are provided below:

[0178] 5A3_Heavy Chain_aa

[0179] QVQLQQSGPELVRPGVSVKISCKVSGYTFTDYAMHWVKQSHAKSLEWIGVISTYSGNTKYN

[0180] QNFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCALGGPRDYFDYWGQGTTLTVSS (SEQ

[0181] ID NO:29)

[0182] F3-4A2-5-HC - 4A2 heavy chain

[0183] QVQLQQSGPELVRPGTSVKVSCKASGYAFTNYLIEWLRQRPGQGLEWIGVINPGSGGTNYN EKFKGKATLTADKSSSTAYMRLGSLTSDDSAVYFCARALRGMDYWGQGTSVTVS (SEQ ID NO:30)

[0184] F3-6H1-6-HC - 6H1 heavy chain

[0185] QVQLQQSGPELVRPGTSVKVSCKASGYAFTNYLIEWLRQRPGQGLEWIGVINPGSGGTNYN EKFKGKATLTADKSSSTAYMRLGSLTSDDSAVYFCARALRGMDYWGQGTSVTVS (SEQ ID NO:31)

[0186] F3-1A3-2-HC - 1A3 heavy chain

[0187] QVQLQQSGPELVRPGTSVKVSCKASGYAFTNYLIEWLRQRPGQGLEWIGVINPGSGGTNYN EKFKGKATLTADKSSSTAYMRLGSLTSDDSAVYFCARALRGMDYWGQGTSVTVS (SEQ ID NO:32)

[0188] F3-1E3-4-HC - 1E3 heavy chain

[0189] QVQLQQSGPELVRPGTSVKVSCKASGYAFTNYLIEWLRQRPGQGLEWIGVINPGSGGTNYN EKFKGKATLTADKSSSTAYMRLGSLTSDDSAVYFCARALRGMDYWGQGTSVTVS (SEQ ID NO:33)

[0190] F3-4H6-2-HC - 4H6 heavy chain

[0191] QIQLVQSGPELKKPGETVKISCKASGYTFTDYSIHWVKQAPGKGLKWMGWINTETGEPTYAD DFKGRFAFSLETSASTAYVQINNLKNEDTATYFCAIDHSFYGDYAMDYWGQGTSVTVS (SEQ ID NO:34)

[0192] F3-1H5-4-HC - 1H5 heavy chain

[0193] EVQLQQSGTVLARPGSSVKMSCKASGYSLTYYWMHWVRQRPGQGLEWIGSIYPRNSDTNY NQKFKGKAKLTAVTSANTAYMELSNLTNEDSAVYYCSREPYYKYDRYFDVWGAGTTVTVS (SEQ ID NO:35)

[0194] F6-1H11-7-HC - 1H11 heavy chain

[0195] EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNGGTTYN QKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGGSTMITTTMTGLYYFAMDYWGQG TSVTVS (SEQ ID NO: 36)

[0196] F5-1D12-1-HC - 1 D12 heavy chain

[0197] EVQLQQSGAELVRPGASVKLSCTASGFNIEDTYIHWVKQRPEQGLEWIGRIDPANGNTEYDP

[0198] KFQGKATITSDTSSNTVFLQLSNLTSEDTAVYFCAQGGLWGHGTVLTVS (SEQ ID NO:37)

[0199] F5-3F7-11-HC - 3F7 heavy chain

[0200] EVQLQQSGAELVKPGASVRLSCTASGFNIKDTYIHWVKQRPQQGLEWIGRIDPANGNKKYD

[0201] PKFQGKATITSDTSSNTAYLQLSSLTSEDTAVYYCAQGGLWGQGTALTVS (SEQ ID NO:38)

[0202] F14-1A9-11-HC - 1A9 heavy chain

[0203] QVQLQQSGAELARPGASVKLSCKASGYTFTTYWLQWVKQRPGQGLEWIGSIYPGDGDTKYI QKFKDKATMTADTSSSTTYMQLSRLASEDSAVYYCARLTFDGGMDYWGQGTSVTVS (SEQ ID NO:39)

[0204] F13-6H8-13-HC - 6H8 heavy chain

[0205] DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMYWVRQAPEKGLEWVAYINSGSSTIYYA

[0206] DTVKGRFTISRDNTKNTLFLQMTSLRSEDTAIYYCARRGLTPVYWYFDVWGAGTTVTVS (SEQ ID NO:40)

[0207] F3-6F12-12-HC - 6F12 heavy chain

[0208] DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMYWVRQAPEKGLEWVAYISYDSNTIYYA DTVKDRFTISRDNPKSTLFLQMTSLRSEDTAMYYCARSGITTVYWYFDVWGAGTTVTVS (SEQ ID NO:41)

[0209] F5-4F9-20-HC - 4F9a heavy chain

[0210] DVQLQESGPGLVKPSQSLSLTCTVTGFSITSDYAWNWIRQFPGNKLEWMGYISYSGITKYNP

[0211] SLRSRISITRDTSKNQFFLQLNSVTPEDTATYYCARFGYGGVWGQGTLVTVS (SEQ ID NO:42)

[0212] F5-4F9-21-HC - 4F9b heavy chain

[0213] EVKLDETGGGLVQPGRPMKLSCVASGFTISDYWMNWVRQSPEKGLEWIAGAIRNKPYNYKT

[0214] YYSDSVKGRFTISRDDSKSGVYLLMNNLRAEDMGIYYCTWQGVLEGYAMDYWGQGTSVTV

[0215] S (SEQ ID NO:43)

[0216] F5-1G1-13-HC - 1G1 heavy chain

[0217] QVQLLQSGPELVKPGASLKISCKASGYSFTGYYIHWVKQSHIKSLEWIGRIYPYSGASNYNQ NFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCARARNLAYWGQGTLVTVS (SEQ ID NO:44)

[0218] MSP_F6-1G3-10 - 1G3 heavy chain

[0219] QVQLQQSGPELVKPGASMKISCKASGYSFTGYSMNWVKQSHGKNLEWIGLINPYDGGTSY NQKFKGKATLTVDKSSSTAYMELISLTSEDSAVYYCARHGAYYRPAWFAYWGQGTLVTVS (SEQ ID NO:45)

[0220] The amino acid sequences of the light chain variable regions of select monoclonal antibodies are provided below:

[0221] 5A3_Light Chain_aa

[0222] QIVLTQSPALMSASPGEKVTMTCSANSSVSYMYWYQHKPRSSPKVWIYLTSNLASGVPARF

[0223] SGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO:46)

[0224] F3-1A3-2-LC - 1A3 light chain

[0225] DWLTQTPLSLPVNIGDQASISCKSPKSLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNRFSGV PGRFTGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPFTFGSGTKLEIKRA (SEQ ID NO:47)

[0226] F3-1E3-4-LC - 1E3 light chain

[0227] DWLTQTPLSLPVNIGDQASISCKSPKSLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNRFSGV PGRFTGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPFTFGSGTKLEIKRA (SEQ ID NO:48)

[0228] F3-1H5-4-LC - 1H5 light chain

[0229] DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLFYWASTRHTGVPDR FTGSGSGTDYTLTISSVQAEDLALYYCQQHDNTPFTFGSGTKLEIKRA (SEQ ID NO:49)

[0230] F3-4A2-5-LC - 4A2 light chain

[0231] DWLTQTPLSLPVNIGDQASISCKSPKSLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNRFSGV PGRFTGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPFTFGSGTKLEIKRA (SEQ ID NQ:50) F3-4H6-2-LC - 4H6 light chain

[0232] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSG

[0233] VPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIKRA (SEQ ID

[0234] NO:51)

[0235] F3-6F12-12-LC - 6F12 light chain

[0236] DIQMTQSPASLSVSEGETVTITCRASENIYSNLAWYQQRQGKSPHLLVYAGTKLADGVPSRF

[0237] SGSGSGTQYSLKINSLQSEDFGSYYCQHFWDTPWTFGGGTKLEIKRA (SEQ ID NO:52)

[0238] F3-6H1-6-LC - 6H1 light chain

[0239] DWLTQTPLSLPVNIGDQASISCKSPKSLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNRFSGV

[0240] PGRFTGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPFTFGSGTKLEIKRA (SEQ ID NO:53)

[0241] F5-1D12-1-LC - 1 D12 light chain

[0242] DIVMIQSQKFMSTSIGDRVSVTCKASQNVGSNVAWYQQKSGQSPKAVIYSASNRYSGVPDR

[0243] FTGRGSGTDFTLTITNVQSEDLAEYFCQQYSTYPFTFGSGTKLEIKRA (SEQ ID NO:54)

[0244] F5-3F7-11-LC - 3F7 light chain

[0245] DIVMMQSQKFMSTSVGDRVSVTCKASQNVGSKVAWYQQKPGQSPKALIYSASSRYSGVPD

[0246] RFTGSGSGTDFTLTISYVQSEDLAEYFCQQYNSYPFTFGSGTKLERKRA (SEQ ID NO:55)

[0247] F5-4F9-20-LC - 4F9a light chain

[0248] DWMTQTPLTLSVTIGQPASISCKSSQSLLDSDGRTYLNWFLQRPGQSPKRLIYLVSKLDFGV

[0249] PDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPQTFGGGSKLEIKRA (SEQ ID NO:56)

[0250] F5-4F9-21-LC - 4F9b light chain

[0251] DIQMTQSPASLSVSVGETVTITCRPSENIYSNLAWYQQKQGKSPQLLVYTATNLADGVPSRF

[0252] SGSGSGTQYSLKINSLQSGDFGNYYCLHFWTTPWTFGGGTKLEIKRA (SEQ ID NO:57)

[0253] F6-1H11-7-LC - 1 H11 light chain

[0254] DIQMTQSPASLSASVGETVTITCRASGNIHNSLAWYQQKQGKSPRLLVYYAKTLADGVPSRF

[0255] SGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPYTFGGGTKLEIKRA (SEQ ID NO:58)

[0256] F13-6H8-13-LC - 6H8 light chain

[0257] DIQMTQSPASLSVSVGETVTITCRASENIYNNLAWFQQKQGKSPQLLVYAATKLADGVPSRF

[0258] SGSGSGTQYSLKINSLQSEDFGSYYCQHFWDIPWTFGGGTKLEIKRA (SEQ ID NO:59)

[0259] F14-1A9-11-LC - 1A9 light chain

[0260] DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLSSRTRKNYLAWYQQKPGQSPKLLIYWASTRE

[0261] SGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNFTFGSGTKLEIKRA (SEQ ID

[0262] NQ:60)

[0263] F6-1G3-10-26-LC - 1G3 light chain

[0264] QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFS

[0265] GSGSGTSYSLTISSMEAEDAATYYCQQWSNTPPTFGGGTKLEIKRA (SEQ ID NO:61)

[0266] F5-1G1-13-16-LC - 1G1 light chain

[0267] DIQMTQSPSSLSASLGERVSLTCRASQDIGADLNWLQQEPDGTIKRLIYATSSLDSGVPKRFS GSRSGSDYSLTISSLESEDFVVYYCLQYASFPYTFGGGTKLEIKRA (SEQ ID NO:62)

[0268] As used herein, “monoclonal antibody 5A3” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:46 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:29.

[0269] As used herein, “monoclonal antibody 4A2” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:47 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NQ:30.

[0270] As used herein, “monoclonal antibody 6H1” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:48 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:31 .

[0271] As used herein, “monoclonal antibody 1A3” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:49 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:32.

[0272] As used herein, “monoclonal antibody 1E3” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NQ:50 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:33.

[0273] As used herein, “monoclonal antibody 4H6” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:51 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:34.

[0274] As used herein, “monoclonal antibody 1H5” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:52 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 35.

[0275] As used herein, “monoclonal antibody 1H11” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:53 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:36.

[0276] As used here, “monoclonal antibody 1 D12” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO: 54 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:37.

[0277] As used herein, “monoclonal antibody 3F7” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:55 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:38.

[0278] As used here, “monoclonal antibody 1A9” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:56 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 39.

[0279] As used herein, “monoclonal antibody 6H8” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:57 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NQ:40.

[0280] As used here, “monoclonal antibody 6F12” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:58 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:41 .

[0281] As used herein, “monoclonal antibody 4F9” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:59 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:42 (4F9a) or to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NQ:60 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:43 (F49b).

[0282] As used herein, “monoclonal antibody 1G1” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:61 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:44. As used herein, “monoclonal antibody 1G3” refers to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:62 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:45.

[0283] Alternatively, “monoclonal antibody 1E3” may refer to the monoclonal antibody produced by the hybridoma cell line deposited at the International Depositary Authority of Canada, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, MB, Canada, R3E 3R2, under accession number XXXXXXXXX on July XX, 2025.

[0284] Alternatively, “monoclonal antibody 1A3” may refer to the monoclonal antibody produced by the hybridoma cell line deposited at the International Depositary Authority of Canada, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, MB, Canada, R3E 3R2, under accession number XXXXXXXXX on July XX, 2025.

[0285] Alternatively, “monoclonal antibody 4A2” may refer to the monoclonal antibody produced by the hybridoma cell line deposited at the International Depositary Authority of Canada, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, MB, Canada, R3E 3R2, under accession number XXXXXXXXX on July XX, 2025.

[0286] Alternatively, “monoclonal antibody 6H1” may refer to the monoclonal antibody produced by the hybridoma cell line deposited at the International Depositary Authority of Canada, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, MB, Canada, R3E 3R2, under accession number XXXXXXXXX on July XX, 2025.

[0287] In some embodiments, the test relies on two monoclonal antibodies that are specific for the glycoprotein of these viruses: 5A3 and 6H8. As discussed below, in one embodiment, 6H8 is conjugated to colloidal gold and serves as the detection antibody, while 5A3 is sprayed on the test line and serves as the capture antibody.

[0288] As discussed herein, our rapid antigen tests rely on unique monoclonal antibodies that are able to detect (at least) the glycoproteins from Ebola virus, Sudan virus, Bundibugyo virus, Tai Forest virus, Bombali virus and Reston virus with high sensitivity and specificity. To our knowledge, this is the only test capable of detecting Ebola virus, Sudan virus, Bundibugyo virus, Tai Forest virus, Bombali virus and Reston virus glycoprotein, potentially making it the best option for a pan-orthoebolavirus diagnostic.

[0289] As will be apparent to those of skill in the art, this rapid antigen test is a useful diagnostic tool at the point-of-care. Specifically, a test capable of rapidly detecting all human pathogenic orthoebolaviruses is very useful in triaging cases and establishing a differential during outbreaks. The armed forces and other government agencies could use this test while the test could also be used at border control points for screening purposes.

[0290] According to an aspect of the invention, there is provided an immunochromatographic lateral flow assay device for detecting Ebola virus glycoprotein, Sudan virus glycoprotein, Bundibugyo virus glycoprotein, Tai Forest virus, Bombali virus and / or Reston virus glycoprotein in a sample comprising:

[0291] (a) a sample pad for receiving the sample to be tested;

[0292] (b) a conjugate release pad pre-treated with a suitable buffer, said conjugate release pad comprising at least one detection antibody for detecting an orthoebolavirus antigen within the sample;

[0293] (c) a nitrocellulose pad comprising at least one paired capture antibody; and

[0294] (d) an absorption pad for absorbing the sample, said device configured such that once a sample is applied, the sample flows in order from (a) to (b) to (c) to (d), wherein the detection antibody and the paired capture antibody is selected from the group consisting of: a monoclonal capture antibody produced by cell line 5A3 and a monoclonal detection antibody produced by cell line 6H8; a monoclonal capture antibody produced by cell line 6F12 and a monoclonal detection antibody produced by cell line 6H8; a monoclonal capture antibody produced by cell line 1 H 11 and a monoclonal detection antibody produced by cell line 6H8; a monoclonal capture antibody produced by cell line 6H8 and a monoclonal detection antibody produced by cell line 6F12; a monoclonal capture antibody produced by cell line 6H8 and a monoclonal detection antibody produced by cell line 4F9; a monoclonal capture antibody produced by cell line 6H8 and a monoclonal detection antibody produced by cell line 1 H11 ; and a monoclonal capture antibody produced by cell line 6H8 and a monoclonal detection antibody produced by cell line 5A3.

[0295] In some embodiments, one monoclonal antibody is 6H8 comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO: 8 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 4.

[0296] In some embodiments, one monoclonal antibody is 5A3 comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO: 6 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 2.

[0297] In some embodiments, the detection monoclonal antibody is 6H8 comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO: 8 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 4 and the monoclonal capture antibody is 5A3 comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO: 6 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 2.

[0298] There are different sample types that can be applied to ILF assays. The source of sample may be a body fluid, for example, but by no means limited to urine, saliva, oral swab, nasopharyngeal swab, whole blood, serum, and cerebrospinal fluid, for example, derived or taken or collected from a human subject. Other samples sources may include wastewater, environmental swabs, virus culture media, or animal-derived samples (i.e. , whole blood, serum, saliva, oral swabs, nasal swabs, rectal swabs, tissue homogenates, etc.). As will be appreciated by one of skill in the art, these samples can all be described as “biological” samples at least by virtue of the presence of biological material within the sample. In some embodiments, the body fluid is chosen based on prevalence of analyte in the sample and ease of sample collection. For the development of our ILF assay, we chose blood as the primary sample type since blood is expected to have very high levels of virus during infection.

[0299] In some embodiments of the invention, there is provided the proviso that the Ebola virus is not EBOV / Makona C05 GP.

[0300] According to another aspect of the invention, there is provided an immunochromatographic lateral flow assay device for detecting Ebola virus glycoprotein, Sudan virus glycoprotein, Bundibugyo virus glycoprotein, Tai Forest virus, Bombali virus and / or Reston virus glycoprotein in a biological sample comprising:

[0301] (a) a sample pad for receiving the biological sample to be tested;

[0302] (b) a conjugate release pad pre-treated with a suitable buffer, said conjugate release pad comprising at least one detection antibody selected from the group consisting of: 1A3; 1E3; 1H5; 4A2; 4H6; 6F12; 6H1 ; 5A3; 1D12; 1G1 ; 4F9; 1G3; 1 H11 ; 6H8; 1A9; and 3F7; for detecting an orthoebolavirus antigen within the biological sample;

[0303] (c) a nitrocellulose pad comprising at least one paired capture antibody selected from the group consisting of 1A3; 1 E3; 1H5; 4A2; 4H6; 6F12; 6H1 ; 5A3; 1 D12 ; 1G1 ; 4F9; 1G3; 1H11 ; 6H8; 1A9; and 3F7; said at least one paired capture antibody being different from the at least one detection antibody but binding to the orthoebolavirus antigen independently and without interfering with the binding of the at least one detection antibody to the antigen

[0304] (d) an absorption pad for absorbing the sample, said device configured such that once a sample is applied, the sample flows in order from (a) to (b) to (c) to (d).

[0305] In some embodiments of the invention, the lateral flow assay device further comprises a second detection antibody that is different from the at least one detection antibody and the at least one capture antibody.

[0306] In some embodiments of the invention, the lateral flow assay device further comprises a second capture antibody that is different from the at least one detection antibody and the at least one capture antibody.

[0307] In some embodiments of the invention, the lateral flow assay device further comprises a second detection antibody that is different from the at least one detection antibody and the at least one capture antibody and a second capture antibody that is different from the at least one detection antibody, the at least one capture antibody and the second detection antibody and is paired to at least the second detection antibody.

[0308] In some embodiments of the invention, there is provided the proviso that the Ebola virus is not EBOV / Makona C05 GP.

[0309] In some embodiments of the invention, there is provided the proviso that the capture and detection antibodies are not 6H8 (capture) paired with 4F9 (detection).

[0310] In some embodiments of the invention, there is provided the proviso that the capture and detection antibodies are not 6F12 as a capture antibody and any one of 5A3, 6H8, 4H6, 6F12, 4F9, and 1A9 as detection antibodies.

[0311] In some embodiments of the invention, there is provided the proviso that the capture and detection antibodies are not 4H6 as a capture antibody and any one of 5A3, 6H8, 4H6, 6F12, 4F9, and 1A9 as detection antibodies. In some embodiments of the invention, there is provided an immunochromatographic lateral flow assay device for Ebola virus glycoprotein detection as set forth above wherein the paired capture and detection antibodies are at least one of 5A3-4F9, 6F12-1A9 and 6H8-1A9.

[0312] In some embodiments of the invention, there is provided an immunochromatographic lateral flow assay device for SUDV glycoprotein detection as set forth above wherein the paired capture and detection antibodies are 6F12-1A9 or 6H8-1A9.

[0313] In some embodiments of the invention, there is provided an immunochromatographic lateral flow assay device for BDBV glycoprotein detection as set forth above wherein the paired capture and detection antibodies are at least one of 4H6-6H8, 6F12-1A9 and 6H8-1A9.

[0314] In some embodiments of the invention, there is provided an immunochromatographic lateral flow assay device for detection of EBOV glycoprotein, SUDV glycoprotein and / or BDBV glycoprotein wherein the paired capture and detection antibodies are 6H8-1A9 and / or 6F12-1A9.

[0315] As will be appreciated by one of skill in the art, there are a number of variables associated with the choice of capture and detection mAbs:

[0316] Specifically, all antibodies need to be tested for their ability to perform as either the capture antibody or as the detection antibody. Not all mAbs are suitable as capture or detection antibodies.

[0317] There are several important aspects that determine whether a given antibody will be suitable as either a capture antibody or a detection antibody in an ILF assay:

[0318] (1) Antibody epitopes and compatibility: The capture antibody and the detection antibody must both be able to bind to the same antigen, independently and without interfering with each other’s binding. At a minimum, capture and detection antibodies must recognize different epitopes. However, the detection antibody must also bind to the antigen in a way that does not occlude (via steric hindrance or the induction of a conformational change) subsequent binding of the capture antibody. This is the primary reason why antibodies cannot necessarily be randomly mixed and matched to generate functional capture and detection antibody pairs.

[0319] (2) Antibody binding affinity: Antibodies incorporated into ILF assays should, ideally, interact with the antigen with high affinity, with a fast association constant and a slow dissociation constant. This means that appropriate antibodies should ideally have an equilibrium dissociation constant (KD) in the nanomolar range, with a lower dissociation constant meaning that the antibody binds more tightly to the antigen. Since not all antibodies possess sufficiently low KD rates, not all antibodies are appropriate for incorporation into an ILF assay.

[0320] (3) Antibody conformation: Antibodies are flexible proteins, and their tertiary structures may be influenced by their environment. Both capture and detection antibodies used in ILF tests must retain their antigen-binding capabilities after being desiccated (following addition to the strip) and rehydrated (following addition of test sample). Moreover, the detection antibody, in particular, must retain a functional conformation and antigen-binding capability after being covalently linked to a signal-producing element or signal-producing moiety, for example but by no means limited to the gold nanospheres. As such, the suitable reporter is a signalproducing element or signal-producing element that is suitable in that it does not interfere with antibody function. As such, this may also be referred to as an antibody-compatible signalproducing moiety. The maintenance of an effective antibody conformation in response to these environmental factors is not necessarily a given.

[0321] The capture antibodies are applied to the nitrocellulose membrane as a thin line. The concentration of mAb on this line needs to yield a strong enough test line without background signal. For example, a 1 mg / mL solution of the capture antibody may be applied as the test line to a nitrocellulose membrane at concentrations of for example, 1 .5 L / cm, 2 pl_ / cm, or 3 pL / cm. As will be appreciated by one of skill in the art, these concentrations are intended for illustrative purposes only and are not necessarily limiting.

[0322] The detection mAbs are conjugated to a suitable reporter, for example, conjugated to 40-nm, 80-nm, and / or 150-nm of gold nanospheres, after which they are sprayed onto the conjugate pad. When the test is run, the successful antibody pairs will result in a visible test line in the presence of the appropriate antigen.

[0323] Other suitable reporters include but are by no means limited to latex beads of various sizes and colours, graphene, colloidal gold, latex spheres, and cellulose nanobeads which can have different sizes (larger size often used to increase sensitivity) and colours (used to multiplex, or could be a characteristic of the size of bead e.g. gold particles) as well as fluorescent dye and horse radish peroxidase conjugations. ?

[0324] The antigen contained in the test sample needs to have enough time to be separated from the other material that may be in the sample (such as cells and large proteins), interact with the detection antibody in the conjugate pad, and then interact with the capture antibody on the membrane. Furthermore, all nonspecific proteins need to be washed away in a timely manner to avoid the appearance of nonspecific signals. A suitable nitrocellulose membrane produces a sharp and intense test line without nonspecific background signal. Examples of different membranes are listed in Table 1. As will be understood by those of skill in the art, there are many different pore sizes available as well as many different treatments such as for example application of surfactants that can be used to prepare nitrocellulose membranes which are suitable for use with the invention and as such are within the scope of the invention.

[0325] The conjugate release pad holds the detection mAb and must maintain the integrity of the antibody upon drying and long-term storage. The conjugate release pad must consistently and uniformly release the antibody completely after wetting with sample and running buffer. To achieve this functionality, the conjugate release pad may be pretreated with conjugate pad buffer, as described above. The pad buffer may comprise, for example, but by no means limited to (1) various buffers and solutions at various pH levels; (2) different proteins, polymers, and detergents, which move through the strip faster than the detection mAb and confer a blocking effect to reduce nonspecific signals; and (3) carbohydrates and sugars, which are essential for ensuring long-term stability and re-solubilization of the detection mAb.

[0326] Suitable sample application pads may be made of for example but by no means limited to cotton, chopped glass with binder or binder-free micro-glass, polyester fibers, blend of fibers and the like. Other suitable materials will be readily apparent to those of skill in the art. In our assay design, we focused on plasma and blood separation pads as we intended to use primarily blood as the test sample. However, sample pads suitable for other body fluids may be used and are within the scope of the invention.

[0327] The absorption pad may be made of for example cotton or another suitably absorbent material. Specifically, the absorption pad has various water absorption capacities to maintain the capillary forces. For example, to promote clearing of the membrane, the absorption pad is required to hold the entire sample and buffer volume and absorb fluids at the rate they are released from the membrane.

[0328] In addition to the conjugate pad buffer, there is a wide range of different assay buffers one can choose to run the assay. These buffers are critical because they (1) move the antigen contained in the test sample across the strip, (2) solubilize the detection mAb, and (3) block the nitrocellulose membrane to reduce nonspecific signals.

[0329] As will be appreciated by those of skill in the art, described above are conditions to be taken into account during the optimization of the assay, that is, optimizing the assay so that a strong signal is always received, even under less than ideal conditions, for example, low sample volume or concentration. However, these optimization steps are considered to be within the ambit of one of skill in the art, that is, these steps represent routine experimentation.

[0330] In some embodiments, the assay buffer is an external component to the strip and is applied from a dropper bottle. The dropper bottle or individual doses of buffer will allow for an easy dispensing of required buffer amount for each test strip.

[0331] It is well known by those of skill in the art that immunoassays can be influenced by human anti-mouse antibodies (HAMA) or rheumatoid factors (RF) as well as other immune complexes or proteins. Accordingly, in some embodiments, a suitable blocker may be added to increase specificity of our assay.

[0332] The invention will now be explained and / or elucidated by way of examples; however, the invention is not necessarily limited to or by the examples.

[0333] The following monoclonal antibodies (mAbs) specific for the glycoprotein (GP) of various filoviruses were generated in-house, as described in Liu et al. J Infect Dis 2023. These antibodies were assessed for their viability as capture / detection pairs in an immunochromatographic lateral flow (ILF) assay to detect orthoebolavirus GP, as described below. mAbs 6H8 and 5A3 can be used in our ILF: 1A3; 1E3; 1H5; 4A2; 4H6; 6F12; 6H1 ; 5A3; 1D12; 1G1 ; 4F9; 1G3; 1H11 ; 6H8; 1A9; and 3F7.

[0334] We obtained recombinant GPs for most of the relevant filoviruses: Ebola virus (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), Reston virus (RESTV), and Marburg virus (MARV). These recombinant GPs lack the transmembrane domain (dTM). We also obtained a variant of each of the EBOV and SUDV GPs, known as soluble GP (sGP). These recombinant proteins were used as antigen to validate our ILF assay and related ELISAs: EBOV GPdTM; SUDV GPdTM; BDBV GPdTM; RESTV GPdTM; MARV GPdTM; EBOV sGP; and SUDV sGP.

[0335] Immunochromatographic Lateral Flow (ILF) Assay

[0336] Upon the generation of antibodies against orthoebolaviruses, we embarked on the first stage of development, which was performing preliminary screening of numerous different mAbs (described below) against multiple different recombinant filoviruses GPs, using an indirect ELISA approach. The aim was to identify capture and detection mAbs.

[0337] The second stage of development consisted of devising a custom lateral flow assay and assay device, which is described in detail above. Approximately 240 antibody pairs were tested against recombinant EBOV GPdTM, SUDV GPdTM, BDBV GPdTM, RESTV GPdTM, MARV GPdTM, EBOV sGP, and SUDV sGP for a total of approximately 1600 individual tests. Detailed mAb screening of a subset of pairs was performed with recombinant proteins via Biolayer interferometry (BLI) (Figure 2 and 3), and with authentic virus (Figure 4 and 7).

[0338] Specifically, as shown in Figure 2, Streptavidin (SA) sensors loaded with biotinylated monoclonal antibodies (mAbs), mirroring gold-conjugated detection mAbs, were exposed to 500 nM recombinant Ebola virus (EBOV, IBT 0501-015), Sudan virus (SUDV, IBT 0502-025) or Bundibugyo virus (BDBV, IBT 0505-015) glycoprotein (GP), followed by exposure to a second, unconjugated mAb, mirroring the capture mAb. The association and dissociation of the second mAb was quantified.

[0339] As will be apparent to one of skill in the art, values in Figure 2 represent the maximum binding signal of the second mAb at the end of the association step, with higher values indicating that binding of the first antibody (i.e., the Detection mAb) does not restrict binding of the second (i.e., the Capture mAb), a critical factor for the functionality of the ILF test. The results revealed that binding of 5A3 to EBOV and BDBV GP with pre-bound 6H8-biotin was the strongest compared to all the other mAb pairs. Binding of 5A3 to SUDV GP with prebound 6H8-biotin was the second strongest, compared to all other mAb pair. Thus, these results demonstrate that 6H8 bound to GP does not obstruct the subsequent binding of 5A3, which demonstrates that 6H8 (as Detection mAb) and 5A3 (as Capture mAb) can be used as a mAb pair for developing a pan-orthoebolavirus ILF test. This test also confirmed that the role of capture and detection antibodies cannot necessarily be reversed.

[0340] As shown in Figure 3, a Sartorius Octet instrument was used to quantify the sequential binding of antibodies to recombinant Ebola virus (EBOV, IBT 0501-015), Sudan virus (SUDV, IBT 0502-025), and Bundibugyo virus (BDBV, IBT 0505-015) glycoprotein (GP). Streptavidin (SA) sensors loaded with biotinylated monoclonal antibodies (mAbs), mirroring gold- conjugated detection mAbs, were exposed to 500 nM recombinant GP, followed by exposure to a second, unconjugated mAb, mirroring the capture mAb. The association and dissociation of the second mAb was quantified.

[0341] This figure graphically represents some of the data in Figure 2, specifically binding of 5A3-Biotin to GP, followed by binding of 6H8 (blue lines), or binding of 6H8-Biotin to GP, followed by binding of 5A3 (orange lines). Y-axis values represent the binding intensity of the second mAb, with higher values indicating that binding of the first antibody does not restrict binding of the second, a critical factor for the functionality of the ILF test. X-axis values represent the time of association and dissociation for the second mAb in seconds (s), with the two steps separated by the dotted line. In this instance, 6H8-Biotin does not restrict binding of 5A3, but 5A3-Biotin does restrict binding of 6H8. Not only do these data suggest that 5A3 (as Capture mAb) and 6H8 (as Detection mAb) make a mAb pair for the pan-orthoebolavirus ILF test, they also suggest that the reciprocal mAb arrangement (i.e. , 5A3 as Detection mAb and 6H8 as Capture mAb) will not work under these conditions, as discussed below. Note that the relatively poor performance of the recombinant SUDV GP is likely due to the inferior quality of the recombinant protein and not due to the antibodies, since later ILF tests performed well with authentic SUDV.

[0342] It is important to appreciate the fact that an ILF test may fail not only because the antibodies used in the test are incompatible (i.e., binding of one antibody interferes with the binding of a second antibody), but also because assay conditions (e.g., reporter conjugate, buffer composition, test substrates) are not optimal. It is essentially impossible to develop an ILF test with incompatible antibodies, but it may be possible to optimize all other test conditions in order to produce a functional test.

[0343] Shown in Figure 4 is the evaluation of six capture-detection mAb pairs in an unoptimized ILF test using 50 pL of Ebola virus / Makona C07 (EBOV; infectious titer of 36,350 median tissue culture infectious dose (TCID50)), Sudan virus / Gulu (SUDV; 6,000 TCID50), Bundibugyo virus / Uganda (BDBV; 5,000 TCID50), Reston virus (RESTV; 2,505 TCID50) or Marburg virus / Angola 325 (MARV; 15,000 TCID50) combined with 50 pL PBS. The test and control lines are indicated with T and C, respectively.

[0344] As can be seen, the 5A3-6H8 mAb pair demonstrated the most positive results, with a clear signal appearing at the test line for EBOV, SUDV, BDBV, and RESTV. Other mAb pairs also demonstrated a signal at the test line: 5A3-4F9, 6F12-1A9, 6H8-1A9 for EBOV; 6F12- 1A9, 6H8-1A9 for SUDV; and 4H6-6H8, 6F12-1A9, 6H8-1A9 for BDBV. No mAb pair detected MARV, which therefore served as a negative control. In all tests, signal was detected at the control line, indicating that the test ran appropriately. Overall, this result agrees with the biolayer interferometry data and indicates that the 5A3-6H8 mAb pair carries the broadest detection spectrum against orthoebolaviruses among the tested mAb pairs, indicating its usefulness as a pan-orthoebolavirus diagnostic tool.

[0345] Shown in Figure 7 are the results of the experiment to determine if the 5A3-6H8 panorthoebolavirus ILF test detects authentic EBOV, SUDV, BDBV, and RESTV in different carriers. In this experiment, Ebola virus / Makona C07 (EBOV; infectious titer of 36,350 media tissue culture infections dose (TCID50)), Sudan virus / Gulu (SUDV; 6,000 TCID50), Bundibugyo virus / Uganda (BDBV; 5,000 TCID50), Reston virus (RESTV; 2,505 TCID50), Marburg virus / Angola 325 (MARV; 15,000 TCID50), Lassa virus (LASV; 50,000 TCID50), or SARS-CoV- 2 (5,000 TCID50) were added to sample running buffer and spiked with 10 pL of plasma or whole blood (donor CE0010253; see Figure 6) per test strip. A total of 100 pL was applied to the 5A3-6H8 pan-orthoebolavirus ILF test. The test and control lines are indicated with T and C, respectively.

[0346] As can be seen, the 5A3-6H8 pan-orthoebolavirus ILF test detected authentic virus in sample running buffer, human plasma, and human whole blood. For EBOV / Makona C07, SUDV / Gulu, BDBV / Uganda, and RESTV, signal at the test line was strongly visible when virus in sample running buffer was applied to the test. Signal was reduced, but not eliminated, for these four viruses when the sample running buffer spiked with human plasma or whole blood. MARV / Angola 325, LASV, and SARS-CoV-2 were not detected at any dilution, and these viruses therefore served as a negative control. In all tests, signal was detected at the control line, indicating that the test ran appropriately.

[0347] We identified a single antibody pair that was capable of specifically detecting RESTV, three antibody pairs that were capable of specifically detecting SUDV GP, four antibody pairs that were capable of specifically detecting BDBV GP and found four antibody pairs that were capable of specifically detecting EBOV GP (Figure 4). Specifically, 6H8-1A9 and 6F12-1A9 recognized EBOV, SUDV and BDBV; 5A3-4F9 recognized EBOV only; and 4H6-6H8 recognized BDBV only.

[0348] Of these tests, we found one antibody pair consisting of capture antibody 5A3 and detection antibody 6H8 that performed well in our inhouse ILF assay for EBOV, SUDV and BDBV recombinant GP and sGP spiked into human blood (Figure 5) and authentic virus (Figure 7).

[0349] Figure 5 shows the results of an experiment to determine if the 5A3-6H8 panorthoebolavirus ILF test detects low concentrations of recombinant glycoproteins. In this experiment, recombinant glycoprotein (GP) or soluble glycoprotein (sGP) from Ebola virus (EBOV, IBT 0501-001 , IBT 0565-001 , respectively), Sudan virus (SUDV, IBT 0502-001 , IBT 0570-001 , respectively), Bundibugyo virus (BDBV, IBT 0505-015), or Marburg virus (MARV, IBT 0506-015) was serially diluted from 500 to 31.2 ng in sample running buffer and spiked with 10 pL per test strip of plasma or whole blood from a human donor (CE0010253; see Figure 6). A total of 100 pL was applied to the ILF test. The test and control lines are indicated with T and C, respectively. As can be seen, the 5A3-6H8 pan-orthoebolavirus ILF test detected recombinant GP from numerous filoviruses in a dose-dependent manner in sample running buffer, human plasma, and human whole blood. For EBOV sGP, signal at the test line was visible down to 62.5 ng of recombinant protein only, and 31.2 ng when plasma or whole blood were added to the test strip. For both EBOV GP, SUDV sGP, and BDBV GP, signal at the test line was visible down to 31.2 ng, even when the sample was spiked with plasma or whole blood. The results from SUDV sGP were particularly robust, suggesting a high degree of sensitivity for this protein. For SUDV GP, signal at the test line was visible down to 125-250 ng of recombinant protein only, and 31.2 ng when plasma or whole blood were added to the test strip. MARV GP was not detected at any dilution, and it therefore served as a negative control. In all tests, signal was detected at the control line, indicating that the test ran appropriately. We believe that the unequal performance of the recombinant sGPs and GPs is due to the recombinant protein and not due to the antibodies, because the ILF test performed well with authentic viruses. Therefore, these results not only show good sensitivity of GP detection by this mAb pair, but they also demonstrate that the pair is capable of detecting orthoebolavirus GPs under stringent conditions, such as in plasma or blood.

[0350] The sensitivity of the 5A3-6H8 pan-orthoebolavirus ILF was demonstrated with authentic / infectious virus via serial dilution and blood samples from historical animal studies (Figure 8 and 9). The 5A3-6H8 pan-orthoebolavirus ILF test strip was unable to detect EBOV variant Makona C05, which was attributed to a single Tryptophan (W) to Arginine (R) mutation in the GP at position 291. EBOV Makona C05 represents only a small portion (0.5%) of EBOV sequences deposited in the NCBI database.

[0351] Figure 8 shows the results of an experiment to determine if the 5A3-6H8 panorthoebolavirus ILF test detects low concentrations of infectious EBOV, SUDV, BDBV, and TAFV. In this experiment, Ebola virus / Makona C07 (EBOV), Sudan virus / Gulu (SUDV), Bundibugyo virus / Uganda (BDBV), Reston virus (RESTV), and Tai Forest virus (TAFV) were serially diluted two-fold in sample running buffer from a starting concentration of 6000 median tissue culture dose (TCID50) or 34,620 TCID50 (EBOV only), and 100 L of each dilution was applied to each test strip. The test and control lines are indicated with T and C, respectively. The cycle threshold (Ct) values from RT-qPCR assays targeting the L gene, or E gene for SARS-CoV-2, in total RNA extracted from whole blood samples are provided for comparison.

[0352] The 5A3-6H8 pan-orthoebolavirus ILF test detected authentic virus in a dosedependent manner. For EBOV / Makona C07, signal at the test line was visible down to 4327 TCID50 of virus, with a faint band at 2164 TCID50. For SUDV / Gulu, BDBV / Uganda, and RESTV, signal at the test line was visible down to at least 750 TCID50. For TAFV, signal at the test line was visible down to 375 TCID50. These results suggest that the pan-orthoebolavirus ILF test (5A3-6H8) can detect low infectious levels of a number of different filoviruses. MARV / Angola 325, LASV, and SARS-CoV-2 were not detected at 6000 TCID50, and these viruses therefore served as a negative control. In all tests, signal was detected at the control line, indicating that the test ran appropriately.

[0353] Figure 9 shows the results of an experiment to determine if the 5A3-6H8 panorthoebolavirus ILF test detects virus in blood samples from experimental animals. In this experiment, whole blood samples from ferrets infected with Ebola virus / Makona C07 (EBOV), Sudan virus / Gulu (SUDV), Bundibugyo virus / Uganda (BDBV), or recombinant EBOV expressing Bombali virus (BOMV) GP instead of EBOV GP (EBOV-BOMV GP) and nonhuman primates (NHPs) infected with EBOV / Makona C07 were tested at various timepoints (i.e., days post infection, dpi) during the course of the infection, as indicated. A total of 10 L whole blood was applied to the test strip and run with 100 L of sample running buffer. The urea concentration of the sample running buffer was doubled for NHPs to avoid false positive results. The cycle threshold (Ct) values from RT-qPCR assays targeting the L gene in total RNA extracted from whole blood samples are provided for comparison. ND, not detected.

[0354] As can be seen, the 5A3-6H8 pan-orthoebolavirus ILF test detected EBOV / Makona C07, SUDV / Gulu, BDBV / Uganda, and BOMV in whole blood samples obtained from experimental animals inoculated with these viruses. EBOV-infected ferret blood samples from two animals (Animal 1 and Animal 2) showed signal at the test line on 5 dpi, but not 2 dpi, while SUDV-infected ferret blood samples from two animals (Animal 3 and Animal 4) showed signal at the test line on 5 dpi, but not 3 dpi. EBOV-infected NHP blood samples from two animals (Animal 9 and Animal 10) showed signal at the test line on 4 and 7 dpi, but not 0 dpi, prior to virus inoculation. Notably, the positive result at 4 dpi was coincident with the first appearance of mild clinical symptoms in these animals. These results demonstrate the conversion of the ILF test from a negative result to a positive result as the virus infection progresses and disease develops. Blood samples from two BDBV-infected ferrets (Animal 5 and Animal 6) and from two ferrets (Animal 7 and Animal 8) infected with EBOV-BOMV GP also displayed signal at the test line on 7 dpi, further support the broad-detection capability of this mAb pair. In all tests, signal was detected at the control line, indicating that the test ran appropriately.

[0355] Figure 10 shows the results of an experiment to determine if the 5A3-6H8 panorthoebolavirus ILF test does not detect EBOV variant Makona C05. (A) Ebola virus / Mayinga (EBOV; infectious dose of 150,000 median tissue culture infectious dose (TCID50)), EBOV / Kikwit (64,700 TCID50), EBOV / Makona C07 (72,700 TCID50), and EBOV / Makona C05 (60,000 TCID50) were diluted 1 :1 with sample running buffer, and 100 pL were applied to the ILF test. (B) A segment of the glycoprotein amino acid sequence alignment is shown for a representative subset of orthoebolaviruses, highlighting the unique polymorphism W291T in EBOV / Makona C05 that presumably prevents detection by the ILF test. (C) This W291R polymorphism is only present in EBOV / Makona C05 variant, which accounts for a total of 14 (0.5%) orthoebolavirus sequences deposited in the NCBI data base.

[0356] The 5A3-6H8 pan-orthoebolavirus ILF test was unable to detect the EBOV / Makona C05 GP, which represents less than 0.5% of publicly available EBOV genome sequences. The inability of this test to recognize EBOV / Makona C05 is likely linked to a Tryptophan (W) to Arginine (R) mutation at position 291 in the glycoprotein, which obstructs binding by one of the two mAbs used in the ILF test. Given the much lower occurrence of EBOV / Makona C05, this analysis indicates that the restriction of pan-orthoebolavirus detection breadth of this ILF test is likely very limited.

[0357] Figure 11 shows the results of an experiment to determine if the 5A3-6H8 panorthoebolavirus ILF test detects SUDV variants Boneface and Gulu, but does not detect MARV variants Angola and Musoke or RAW. (A) Sudan virus (SUDV) variants Boneface (infectious titer of 6,000 median tissue culture infectious dose (TCID50)) and Gulu (6,000 TCID50) were diluted 1 :1 with 2X sample running buffer, and 100 pL were applied to the ILF test. (B) Marburg virus (MARV) variants Angola 325 (158,000 TCID50) and Musoke (28,100 TCID50), as well as Ravn virus (RAW; 232,200 TCID50) were diluted 1 :1 with 2X sample running buffer, and 100 pL were applied to the ILF test.

[0358] As can be seen, the 5A3-6H8 pan-orthoebolavirus ILF test was able to detect the SUDV glycoprotein from variants Boneface and Gulu. High viral titres of MARV and RAW were negative. In all tests, signal was detected at the control line, indicating that the test ran appropriately. These results further confirm the fact that this ILF test is specific to orthoebolaviruses and does not detect orthomarburgviruses.

[0359] Figure 1 depicts the general setup of the assay strips. The ILF assay used in this example used the following reagents, although other suitable reagents and / or combinations thereof may be used and are within the scope of the invention.

[0360] The capture mAb: 5A3 diluted to 1 mg / ml with 1x PBS, applied as the test line to the nitrocellulose membrane using a CAMAG Linomat 5 at concentrations of 1.5 pL / cm, 2 pL / cm, or 3 pL / cm.

[0361] The detection mAb: 6H8 conjugated to 80-nm gold nanospheres (Nanocomposix), applied to the conjugate release pad using a CAMAG Linomat 5 at a concentration of 10 pL / cm after blocking with antibody diluent (2% BSA, 1% Tween in 5 mM sodium phosphate).

[0362] The control antibody: anti-mouse IgG whole molecule antibody produced in goat (Sigma, M8542-1mg) diluted to 1 mg / ml with 0.135 M Sodium Chloride, applied as the control line to the nitrocellulose membrane using a CAMAG Linomat 5 at a concentration of 1 pL / cm.

[0363] The conjugate pad buffer (10% sucrose, 5% trehalose, 0.5% BSA, 0.1% Triton X-100 in 1X PBS), in which the conjugate pad is immersed.

[0364] The sample running buffer: 40 mM Tris (Roche 11814273001 , Lot: 0609LCK6AA), 300 mM NaCI (BDH 9286, Lot: 22C1556448), 500 mM urea (Fisher BP169-212, Lot 147598A), 0.5% bovine serum albumin (VWR 0332, Lot: 2161956244), 0.5% Tween 80 (Sigma 59929, Lot BCBN2111 V), pH 7.5, 7.75, 8.0, 8.25, or 8.5

[0365] To assemble the ILF test in the examples, the detection mAb is conjugated to 80-nm gold nanospheres as per manufacturer's instructions. The conjugate release pad is treated with conjugate pad buffer and dried (then stored at <20% relative humidity), after which the detection mAb is applied and the conjugate pad is dried again, this time in a forced-air convection oven for 1 h at 37°C. The capture and control antibody are diluted and applied to the nitrocellulose membrane and dried. Following the preparation of all physical components, the test strips are assembled by laminating each component onto a backing card in the following order: (1) nitrocellulose membrane, (2) conjugate pad, (3) sample application pad, (4) absorption pad. Once assembled, the test strips are cut to size and stored <20% relative humidity at ambient temperature until use.

[0366] At the time of test, 10 pL of human blood or plasma spiked with antigen is applied to the sample pad. This is then followed with 100 pL of running buffer. The test is allowed to develop for 15 to 30 minutes, after which the sample pad is clipped off the strip to stop the development and / or an image of the strip is captured. Alternatively, the results are recorded on a spread sheet. While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

[0367] Table 1: A List of Some Membranes and Their Characteristics

Claims

CLAIMS1. An immunochromatographic lateral flow assay device for detecting Ebola virus glycoprotein, Sudan virus glycoprotein, Bundibugyo virus glycoprotein, Tai Forest virus glycoprotein, Bombali virus glycoprotein and / or Reston virus glycoprotein in a sample comprising:(a) a sample pad for receiving the sample to be tested;(b) a conjugate release pad pre-treated with a suitable buffer, said conjugate release pad comprising at least one detection antibody for detecting an orthoebolavirus antigen within the sample;(c) a nitrocellulose pad comprising at least one paired capture antibody; and(d) an absorption pad for absorbing the sample, said device configured such that once a sample is applied, the sample flows in order from (a) to (b) to (c) to (d), wherein the detection antibody and the paired capture antibody is selected from the group consisting of: a monoclonal capture antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:46 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:29 (5A3) and a monoclonal detection antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:57 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NQ:40 (6H8); a monoclonal capture antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:58 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:41 (6F12) and a monoclonal detection antibody 6H8; a monoclonal capture antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:53 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:36 (1 H11) and a monoclonal detection antibody 6H8; a monoclonal capture antibody 6H8 and a monoclonal detection antibody produced by cell line 6F12; a monoclonal capture antibody 6H8 and a monoclonal detection antibody comprisinga light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:59 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:42 (4F9a) or to a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NQ:60 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:43 (4F9b); a monoclonal capture antibody 6H8 and a monoclonal detection antibody produced by cell line 1H11; and a monoclonal capture antibody 6H8 and a monoclonal detection antibody 5A3.

2. The immunochromatographic lateral flow assay device according to claim 1 wherein the monoclonal antibody is 6H8 comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO: 57 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 40.

3. The immunochromatographic lateral flow assay device according to claim 1 wherein the monoclonal antibody is 5A3 comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:46 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 29.

4. The immunochromatographic lateral flow assay device according to claim 1 wherein the detection monoclonal antibody is 6H8 comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:57 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NQ:40 and the monoclonal capture antibody is 5A3 comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:46 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:29.

5. The immunochromatographic lateral flow assay device according to claim 1 wherein the sample to be tested is a body fluid.

6. The immunochromatographic lateral flow assay device according to claim 5 wherein the body fluid is urine, saliva, oral swab, nasopharyngeal swab, whole blood, serum, and cerebrospinal fluid.

7. The immunochromatographic lateral flow assay device according to claim 1wherein the capture antibodies are applied to the nitrocellulose pad as a thin line.

8. The immunochromatographic lateral flow assay device according to claim 7 wherein the capture antibodies are applied to the nitrocellulose pad at concentrations of 1.5 pL / cm to 3 pL / cm based on a 1 mg / mL solution.

9. The immunochromatographic lateral flow assay device according to claim 1 wherein the detection monoclonal antibodies are conjugated to a suitable reporter.

10. The immunochromatographic lateral flow assay device according to claim 9 wherein the suitable reporter is gold nanospheres, latex beads, graphene, colloidal gold, latex spheres, cellulose nanobeads, fluorescent dye conjugations or horse radish peroxidase conjugations.

11. The immunochromatographic lateral flow assay device according to claim 1 wherein the sample is wastewater, environmental swabs, virus culture media, or animal- derived samples.

12. The immunochromatographic lateral flow assay device according to claim 1 wherein the lateral flow assay device further comprises a second detection antibody that is different from the at least one detection antibody and the at least one capture antibody.

13. The immunochromatographic lateral flow assay device according to claim 1 wherein the lateral flow assay device further comprises a second capture antibody that is different from the at least one detection antibody and the at least one capture antibody.

14. The immunochromatographic lateral flow assay device according to claim 1 wherein the lateral flow assay device further comprises a second detection antibody that is different from the at least one detection antibody and the at least one capture antibody and a second capture antibody that is different from the at least one detection antibody, the at least one capture antibody and the second detection antibody and is paired to at least the second detection antibody.

15. An immunochromatographic lateral flow assay device for detecting Ebola virus glycoprotein, Sudan virus glycoprotein, Bundibugyo virus glycoprotein, Tai Forest virus glycoprotein, Bombali virus glycoprotein and / or Reston virus glycoprotein in a sample comprising:(a) a sample pad for receiving the sample to be tested;(b) a conjugate release pad pre-treated with a suitable buffer, said conjugate release pad comprising at least one detection antibody selected from the group consisting of: a monoclonal antibody comprising a light chain comprising the light chain variableregion amino acid sequence as set forth in SEQ ID NO:49 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:32 (1A3); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NQ:50 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:33 (1E3); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:52 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 35 (1H5); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:47 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NQ:30 (4A2); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:51 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:34 (4H6); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:58 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:41 (6F12); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:48 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:31 (6H1); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:46 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:29 (5A3); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO: 54 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:37 (1D12); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:61 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:44 (1G1); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:59 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:42 (4F9a) or to a monoclonal antibody comprising a light chain comprising the light chain variable region aminoacid sequence as set forth in SEQ ID NO:60 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:43 (4F9b); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:62 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:45 (1G3); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:53 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:36 (1H11); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:57 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NQ:40 (6H8); a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:56 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 39 (1A9); and a monoclonal antibody comprising a light chain comprising the light chain variable region amino acid sequence as set forth in SEQ ID NO:55 and a heavy chain comprising the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:38 (3F7); for detecting an orthoebolavirus antigen within the sample;(c) a nitrocellulose pad comprising at least one paired capture antibody selected from the group consisting of 1A3; 1 E3; 1 H5; 4A2; 4H6; 6F12; 6H1; 5A3; 1D12 ; 1G1; 4F9; 1G3; 1 H11 ; 6H8; 1A9; and 3F7; said at least one paired capture antibody being different from the at least one detection antibody but binding to the orthoebolavirus antigen independently and without interfering with the binding of the at least one detection antibody to the antigen(d) an absorption pad for absorbing the sample, said device configured such that once a sample is applied, the sample flows in order from (a) to (b) to (c) to (d).

16. The immunochromatographic lateral flow assay device according to claim 15 wherein the sample to be tested is a body fluid.

17. The immunochromatographic lateral flow assay device according to claim 16 wherein the body fluid is urine, saliva, oral swab, nasopharyngeal swab, whole blood, serum, and cerebrospinal fluid.

18. The immunochromatographic lateral flow assay device according to claim 15 wherein the sample is wastewater, environmental swabs, virus culture media, or animal-derived samples.

19. The immunochromatographic lateral flow assay device according to claim 15 wherein the capture antibodies are applied to the nitrocellulose pad as a thin line.

20. The immunochromatographic lateral flow assay device according to claim 19 wherein the capture antibodies are applied to the nitrocellulose pad at concentrations of 1.5 pL / cm to 3 pL / cm based on a 1 mg / mL solution.

21. The immunochromatographic lateral flow assay device according to claim 15 wherein the detection monoclonal antibodies are conjugated to a suitable reporter.

22. The immunochromatographic lateral flow assay device according to claim 21 wherein the suitable reporter is gold nanospheres, latex beads, graphene, colloidal gold, latex spheres, cellulose nanobeads, fluorescent dye conjugations or horse radish peroxidase conjugations.

23. The immunochromatographic lateral flow assay device according to claim 15 wherein the lateral flow assay device further comprises a second detection antibody that is different from the at least one detection antibody and the at least one capture antibody.

24. The immunochromatographic lateral flow assay device according to claim 15 wherein the lateral flow assay device further comprises a second capture antibody that is different from the at least one detection antibody and the at least one capture antibody.

25. The immunochromatographic lateral flow assay device according to claim 15 wherein the lateral flow assay device further comprises a second detection antibody that is different from the at least one detection antibody and the at least one capture antibody and a second capture antibody that is different from the at least one detection antibody, the at least one capture antibody and the second detection antibody and is paired to at least the second detection antibody.

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