Neutralizing antibody immunoassays
AssigneeInBios International, Inc.
InventorsGregory Ireton, Syamal Raychaudhuri, James Needham, Dindo Reyes
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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.
Claims17
An enzyme-linked immunosorbent assay (ELISA) method of detecting antibodies that are neutralizing for SARS-CoV-2 Spike protein binding to human angiotensin-converting enzyme 2 (ACE2), the method comprising:
- combining full-length SARS-CoV-2 Spike protein ectodomain with a strep tag protein label at one terminus and an anti-strep tag protein label antibody bound to the strep tag with a sample comprising antibodies to form a mixture;
- incubating the mixture with a human ACE2 protein ectodomain bound to a substrate for a length of time sufficient to allow the full-length SARS-CoV-2 Spike protein to bind the human ACE2 protein ectodomain;
- incubating the substrate with a detection antibody that specifically binds a constant region of the anti-strep tag protein label antibody and comprises a detection label for a length of time sufficient for the detection antibody to bind to the anti-strep tag protein label antibody;
- washing the substrate to remove SARS-CoV-2 Spike protein ectodomain with a strep tag protein label at one terminus, anti-strep tag protein label antibody, and detection antibody not bound to the human ACE2 protein ectodomain;
- incubating the substrate with an enzymatic substrate;
- and
- measuring the amount of anti-strep tag protein label antibody associated with the substrate as compared to a negative control by measuring an amount of the detection label;
- wherein a reduced amount of detection label as compared to the negative control indicates the presence of antibodies in the sample that are neutralizing for SARS-CoV-2 Spike protein binding to human ACE2.
An enzyme-linked immunosorbent assay (ELISA) method of detecting antibodies that are neutralizing for SARS-CoV-2 Spike protein binding to human angiotensin-converting enzyme 2 (ACE2), the method comprising:
- combining full-length SARS-CoV-2 Spike protein ectodomain with a strep tag protein label at one terminus and an anti-strep tag protein label antibody bound to the strep tag with a sample comprising antibodies to form a mixture;
- incubating the mixture with a human ACE2 protein ectodomain bound to a substrate for a length of time sufficient to allow the full-length SARS-CoV-2 Spike protein to bind the human ACE2 protein ectodomain;
- incubating the substrate with a detection antibody that specifically binds a constant region of the anti-strep tag protein label antibody and comprises an enzymatic detection label for a length of time sufficient for the detection antibody to bind to the anti-strep tag protein label antibody;
- washing the substrate to remove SARS-CoV-2 Spike protein ectodomain with a strep tag protein at one terminus, anti-strep tag protein label antibody, and detection antibody not bound to the human ACE2 protein ectodomain;
- incubating the substrate with an enzymatic substrate for a length of time sufficient for the enzymatic detection label to catalyze a reaction with the enzymatic substrate to produce a fluorescent or color reaction product;
- and
- measuring the amount of anti-strep tag protein label antibody associated with the substrate as compared to a negative control by measuring an amount of a fluorescent or color reaction product;
- wherein a reduced amount of fluorescent or color reaction product as compared to the negative control indicates the presence of antibodies in the sample that are neutralizing for SARS-CoV-2 Spike protein binding to human ACE2.
Dependencies are inferred from the claim text.