Neutralizing antibody immunoassays
Inventors
Ireton, Gregory • Raychaudhuri, Syamal • Needham, James • Reyes, Dindo
Assignees
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Publication Number
US-11693011-B2
Publication Date
2023-07-04
Expiration Date
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.
Core Innovation
The invention provides an enzyme-linked immunosorbent assay method for detecting antibodies that are neutralizing for SARS-CoV-2 Spike protein binding to human angiotensin-converting enzyme 2 (ACE2). Neutralization is indicated without performing live virus or pseudovirus neutralization assays, by measuring inhibition of Spike binding to ACE2 using a labeled full-length SARS-CoV-2 Spike protein ectodomain and an anti-strep tag protein label detection system tied to a negative control.
The method forms a mixture of full-length Spike ectodomain with a strep tag protein label and an anti-strep tag protein label antibody, incubates the mixture with human ACE2 protein ectodomain bound to a substrate, and then measures an enzyme, color, or fluorescent signal associated with the anti-strep tag protein label antibody. The assay includes washing to remove unbound components, incubating with an enzymatic substrate, and measuring the amount of detection label as compared to a negative control.
The disclosed platform is further extended to lateral flow assay formats using nitrocellulose membranes with a gold nanoparticle labeled Spike reagent and ACE2 test/control line targets. Neutralization is determined by measuring inhibition reflected in test/control line intensity ratio and applying ROC-derived cutoffs, including threshold-based interpretations, with validation documented by correlation to plaque reduction neutralization tests and by cross-reactivity and performance/robustness results.
Claims Coverage
The partial content provides two independent ELISA claims. Across the independent claims, the detection of neutralizing antibodies relies on an ELISA inhibition format with a strep tag protein label system, ACE2 ectodomain binding on a substrate, and signal reduction relative to a negative control, where the enzymatic detection is read either as detection label amount or as fluorescent or color reaction product.
Strep tag-labeled full-length Spike inhibition ELISA with detection antibody binding anti-strep label constant region
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; 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; washing the substrate; incubating the substrate with an enzymatic substrate; and measuring an 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 that are neutralizing for SARS-CoV-2 Spike protein binding to human ACE2.
Fluorescent or color enzymatic product ELISA inhibition measured versus negative control
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; 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; washing the substrate; incubating the substrate with an enzymatic substrate to produce a fluorescent or color reaction product; and measuring an 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.
Across the two independent ELISA claims, the inventive concept is an inhibition-based ELISA in which neutralizing antibodies reduce an enzyme-associated detection signal generated through a detection antibody that binds a constant region of the anti-strep tag protein label antibody. The claims require full-length SARS-CoV-2 Spike ectodomain labeled with a strep tag, incubation with ACE2 ectodomain bound to a substrate, washing, enzymatic substrate incubation, and comparison to a negative control to indicate neutralizing activity.
Stated Advantages
Avoids live virus or pseudovirus neutralization assays.
Provides detection of antibodies that are neutralizing for SARS-CoV-2 Spike protein binding to human ACE2 using inhibition of Spike binding measured by ELISA signal reduction relative to a negative control.
Provides quantitative interpretation via signal inhibition percent and threshold-based classification as described in dependent claims.
Documented Applications
Detecting SARS-CoV-2 neutralizing antibodies that inhibit SARS-CoV-2 Spike protein binding to human ACE2 using an ELISA format.
Detecting neutralizing antibodies using a lateral flow assay format with gold-labeled Spike and ACE2 test/control lines, interpreted using smartphone or AI reader signals based on test/control line intensity ratio and ROC-derived cutoffs.
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