Neutralizing antibody immunoassays

Abstract

The present disclosure provides compositions and methods for detecting the presence of neutralizing antibodies in a sample. Unlike conventional assays, the methods provided herein do not require the use of live virus or virus pseudoparticles to identify neutralizing antibodies.

1 - 30 . (canceled) 
     
     
         31 . A lateral flow assay comprising: a nitrocellulose membrane; a sample pad resting on the nitrocellulose membrane at a sample end; a conjugate pad resting on the nitrocellulose membrane in a direction of sample flow after the sample pad and comprising a labeled SARs-CoV-2 spike protein and a labeled control antibody that specifically binds a control target protein; a bridge pad resting on the nitrocellulose membrane in a direction of sample flow after the conjugate pad; a test line and a control line on the nitrocellulose membrane both in a direction of sample flow after the bridge pad, wherein the test line comprises a human ACE2 protein eciodomain bound to the nitrocellulose membrane and the control line comprises the control target protein bound to the nitrocellulose membrane; and an absorbent pad in a direction of sample flow after the conjugate pad. 
     
     
         32 . The assay of  claim 31 , wherein the spike protein comprises full-length spike protein ectodomain. 
     
     
         33 . The assay of  claim 32 . wherein the full-length spike protein ectodomain comprises the amino acid sequence according to SEQ ID NO: 1. 
     
     
         34 . The assay of  claim 31 , further comprising a lateral flow assay cassette in which the nitrocellulose membrane; pads, and test lines are housed. 
     
     
         35 . A method of detecting antibodies that are neutralizing for SARS-CoV-2 spike protein binding to human ACE2, the method comprising: (a) placing a sample comprising antibodies on the sample pad of an assay according to claim  1 ; (b) allowing the sample to remain on the assay for a period of time sufficient for the sample to flow over the test line and the control line and for SARS-CoV-2 spike protein in the sample to bind to human ACE2 in the test line; and (c) viewing the test line and the control line to determine if the control line is visible, indicating a valid assay, and if the test line is visible, indicating an absence or limited amount of neutralizing antibodies in the sample. 
     
     
         36 . The method of  claim 35 , further comprising placing a chase buffer on the sample pad after the sample. 
     
     
         37 . The method of  claim 35 , further comprising determining the intensity of the test line to provide a semi-quantitative of quantitative measure of neutralizing antibodies in the sample. 
     
     
         38 . The method of  claim 37 , comprising determining the intensity of the test line using a reader. 
     
     
         39 . The method of  claim 38 , wherein the reader comprises a camera and a smartphone app comprising an artificial intelligence component. 
     
     
         40 . The method of  claim 39 , further comprising displaying a neutralizing antibody result of the method to a user. 
     
     
         41 . The method of  claim 39 , further comprising providing a code corresponding to a neutralizing antibody result of the method to a second reader. 
     
     
         42 . The method of  claim 38 , wherein determining the intensity of the test line using a reader comprises: obtaining one or more first images of a lateral flow assay cassette used to perform the method; aligning the cassette by overlaying a semi-transparent reference of the lateral flow assay device on the one or more first images; presenting the one or more overlaid images to a user in real time as the one or more first images are being obtained; employing a first artificial intelligence mechanism to determine a position of the lateral flow assay cassette in the one or more first images relative to the transparent reference; responsive to the position of the lateral flow assay cassette being in an acceptable position relative to the semi-transparent reference in at least one of the one or more first images: capturing a second image of the lateral flow assay cassette; employing a second artificial intelligence mechanism on the second image to determine if a result of the lateral flow assay cassette is valid; responsive to the result of the lateral flow assay cassette being invalid, presenting an invalid-test-result notification to the user; responsive to the result of the lateral flow assay cassette being valid, employing a third artificial intelligence mechanism on the second image to determine the presence or absence of and/or semi -quantitative or quantitative amount of neutralizing antibody in the sample; and presenting the presence or absence of and/or semi-quantitative or quantitative amount of neutralizing antibody in the sample result to the user. 
     
     
         43 . The method of  claim 42 , wherein employing a third artificial intelligence mechanisin on the second image comprises: determining the intensity of the control line by analytical methods comprising integrating grayscale images around the control line and determining the peak value present in the control line; determining the intensity of the test line by analytical methods comprising integrating grayscale images around the test line and determining the peak value present in the test line; determining the ratio of the test line peak value to the control line peak value; and using the determined ratio to present the presence or absence of and/or the semi-quantitative or quantitative amount of neutralizing antibody in the sample result to the user. 
     
     
         44 . A kit for detection of antibodies that are neutralizing for SARS-CoV-2 Spike protein binding to human ACE2 the kit comprising an assay according to  claim 31 . 
     
     
         45 . The kit of  claim 44 , thither comprising one or more of a sample buffer, a chase buffer, a code identifying a kit type or lot number, a code identifying the sample, a sample collection device, or instructions for installing a smartphone app suitable to cause a smartphone to act as a reader.