Serological assays for sars-cov-2

Abstract

Provided herein is a test system comprising severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) antigen, SARS-CoV-2 S2 antigen, and binding moieties that specifically bind to human IgG, human IgA, and human IgM. Also provided are methods of detecting SARS-CoV-2 antibodies in a sample using the test system.

1 . A test system comprising:
 a first surface comprising a first antigen comprising an amino acid sequence of at least 50 amino acids in length and less than 300 amino acids in length that has at least 99% identity to the amino acid sequence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) antigen; and   a second surface comprising a second antigen comprising an amino acid sequence of at least 100 amino acids in length and less than 600 amino acids in length that has at least 99% identity to the amino acid sequence of the SARS-CoV-2 S2 antigen,   one or more first binding moieties that specifically bind to a constant region of human IgG, one or more second binding moieties that specifically bind to a constant region of human IgA and one or more third binding moieties that specifically bind to a constant region of human IgM.   
     
     
         2 . The test system of  claim 1 , wherein the first antigen comprises the amino acid sequence of SARS-CoV-2 RBD antigen. 
     
     
         3 . The test system of  claim 1  or  2 , wherein the second antigen comprises the amino acid sequence of SARS-CoV-2 S2 antigen. 
     
     
         4 . The test system of any one of  claims 1 - 3 , wherein the binding moieties are antibodies. 
     
     
         5 . The test system of any one of  claims 1 - 4 , wherein the first, second and third binding moieties further comprise a detectable moiety. 
     
     
         6 . The test system of any one of  claims 1 - 5 , wherein the first surface is substantially free from full-length SARS-CoV-2 spike protein. 
     
     
         7 . The test system of any one of  claims 1 - 5 , wherein the first surface is substantially free from a fragment of full-length SARS-CoV-2 S1 protein greater than 300 amino acids in length or a fragment of full-length SARS-CoV-2 S1 protein that does not comprise the RBD. 
     
     
         8 . The test system of any one of  claims 1 - 7 , wherein the second surface is substantially free from full-length SARS-CoV-2 spike protein. 
     
     
         9 . The test system of any one of  claims 1 - 7 , wherein the second surface is substantially free from full-length SARS-CoV-2 S1 protein. 
     
     
         10 . The test system of any one of  claims 5 - 9 , wherein the detectable moiety is a chromogenic label. 
     
     
         11 . The test system of  claim 10 , wherein the chromogenic label comprises horseradish peroxidase (HRP). 
     
     
         12 . The test system of  claim 11 , wherein incubation of the first, second or third binding moieties with an HRP substrate produces a colorimetric signal. 
     
     
         13 . The test system of any one of  claims 1 - 12 , wherein the first surface is contained within a first well and the second surface is contained in a second well. 
     
     
         14 . A test system comprising:
 a first well comprising:
 an immobilized first binding moiety that specifically binds to a constant region of human IgG, an immobilized second binding moiety that specifically binds to a constant region of human IgA and an immobilized third binding moiety that specifically binds to a constant region of human IgM; 
 a first biological sample from a host; and 
 a first antigen comprising an amino acid sequence of at least 50 amino acids in length and less than 300 amino acids in length that has at least 99% identity to the amino acid sequence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) antigen, wherein the first antigen comprises a detectable moiety; 
 and 
   a second well comprising:
 an immobilized first binding moiety that specifically binds to a constant region of human IgG, an immobilized second binding moiety that specifically binds to a constant region of human IgA and an immobilized third binding moiety that specifically binds to a constant region of human IgM; 
 a second biological sample from the host; and 
 a second antigen an amino acid sequence of at least 100 amino acids in length and less than 600 amino acids in length that has at least 99% identity to the amino acid sequence of the SARS-CoV-2 S2 antigen, wherein the second antigen comprises a detectable moiety. 
   
     
     
         15 . A kit, comprising the test system of any one of  claims 1 - 14 , comprising:
 the first antigen; the second antigen;   and   the first binding moiety, the second binding moiety, and the third binding moiety, in separate containers.   
     
     
         16 . A method for detecting the presence of host antigen-specific antibodies that specifically bind SARS-CoV-2, the method comprising the steps of:
 1) exposing a first biological sample from the host to a first surface comprising a first antigen comprising an amino acid sequence of at least 50 amino acids in length and less than 300 amino acids in length that has at least 99% identity to the amino acid sequence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) antigen;   2) incubating the first surface with a first binding moiety that specifically binds to a constant region of human IgG, a second binding moiety that specifically binds to a constant region of human IgA and a third binding moiety that specifically binds to a constant region of human IgM;   3) detecting binding of one or more of the first, second and third binding moieties at the first surface to host antigen-specific antibodies, which generates a first signal;   4) exposing a second biological sample from the host to a second surface comprising a second antigen an amino acid sequence of at least 100 amino acids in length and less than 600 amino acids in length that has at least 99% identity to the amino acid sequence of the SARS-CoV-2 S2 antigen;   5) incubating the first surface with a fourth binding moiety that specifically binds to a constant region of human IgG, a fifth binding moiety that specifically binds to a constant region of human IgA and a sixth binding moiety that specifically binds to a constant region of human IgM; and   6) detecting binding of one or more of the fourth, fifth and sixth binding moieties at the second surface to host antigen-specific antibodies, which generates a second signal,   thereby detecting the presence of host antigen-specific antibodies that specifically bind SARS-CoV-2.   
     
     
         17 . The method of  claim 16 , wherein the first antigen comprises the amino acid sequence of SARS-CoV-2 RBD antigen. 
     
     
         18 . The method of  claim 16  or  17 , wherein the second antigen comprises the amino acid sequence of SARS-CoV-2 S2 antigen. 
     
     
         19 . The method of any one of  claims 16 - 18 , wherein the binding moieties are antibodies. 
     
     
         20 . The method of any one of  claims 16 - 19 , wherein the first, second, third, fourth, fifth and sixth binding moieties further comprise a detectable moiety. 
     
     
         21 . The method of any one of  claims 16 - 19 , wherein the first surface is substantially free from full-length SARS-CoV-2 spike protein. 
     
     
         22 . The method of any one of  claims 16 - 19 , wherein the first surface is substantially free from a fragment of full-length SARS-CoV-2 S1 protein greater than 300 amino acids in length or a fragment of full-length SARS-CoV-2 S1 protein that does not comprise the RBD. 
     
     
         23 . The method of any one of  claims 16 - 22 , wherein the second surface is substantially free from full-length SARS-CoV-2 spike protein. 
     
     
         24 . The method of any one of  claims 16 - 22 , wherein the second surface is substantially free from full-length SARS-CoV-2 S1 protein. 
     
     
         25 . The method of any one of  claims 16 - 24 , wherein the first binding moiety is the same as the fourth binding moiety, the second binding moiety is the same as the fifth binding moiety and/or the third binding moiety is the same as the sixth binding moiety. 
     
     
         26 . The method of any one of  claims 16 - 25 , wherein the biological sample is selected from the group consisting of serum, blood, plasma, sputum, urine, semen, mucous, sweat and tears. 
     
     
         27 . The method of any one of  claims 20 - 26 , wherein the detectable moiety is a chromogenic label. 
     
     
         28 . The method of  claim 27 , wherein the chromogenic label comprises horseradish peroxidase (HRP). 
     
     
         29 . The method of  claim 28 , wherein incubation of the first, second and third binding moieties with an HRP substrate produces a colorimetric signal. 
     
     
         30 . The method of any one of  claims 16 - 29 , wherein:
 a first signal value that is at least five standard deviations above a negative control sample indicates a first positive result;   a first signal value that is at least three standard deviations but less than five standard deviations above a negative control sample indicates a first indeterminate result;   a first signal value that is less than three standard deviations above a negative control sample indicates a first negative result;   a second signal value that is at least five standard deviations above a negative control sample indicates a second positive result;   a second signal value that is at least three standard deviations but less than five standard deviations above a negative control sample indicates a second indeterminate result; and   a second signal value that is less than three standard deviations above a negative control sample indicates a second negative result.   
     
     
         31 . The method of  claim 30 , wherein a first positive result and a second positive result indicates the presence of host antigen-specific antibodies. 
     
     
         32 . The method of  claim 30 , wherein a first positive result and a second indeterminate result indicates the presence of host antigen-specific antibodies. 
     
     
         33 . The method of  claim 30 , wherein a first positive result and a second negative result indicates an absence of host antigen-specific antibodies. 
     
     
         34 . The method of  claim 30 , wherein a first indeterminate result and a second negative result indicates an absence of host antigen-specific antibodies. 
     
     
         35 . The method of any one of  claims 31 - 34 , wherein the host antigen-specific antibodies are neutralizing host antigen-specific antibodies that specifically bind SARS-CoV-2. 
     
     
         36 . The method of any one of  claims 30 - 35 , wherein if there is no first positive result, steps 4-6 are not performed.