Assays for detecting SARS-CoV-2
Inventors
Blyn, Lawrence B. • Ji, Mijung • Kovacs, Stephen • Muerhoff, Anthony S. • Huth, Stacey P. • Buenning, Carsten • Xin, Tao • Roberts, Donabel • Kim, Sung Hee • Park, Sang Yong • Ziemann, Robert N.
Assignees
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Publication Number
US-11988671-B2
Publication Date
2024-05-21
Expiration Date
Abstract
The present disclosure relates to methods of detecting a protein from the SARS-CoV-2 virus, or a fragment thereof, in a sample obtained from a subject using a first antibody or antigen-binding fragment thereof that binds to a protein from the SARS-CoV-2 virus, or a fragment thereof, and a second antibody or antigen-binding fragment thereof which binds to a protein from the SARS-CoV-2 virus, or a fragment thereof.
Core Innovation
The invention relates to an immunoassay for detecting SARS-CoV-2 virus in a sample obtained from a subject, where the immunoassay uses a nucleocapsid (N) protein-specific first antibody or antigen-binding fragment and a conjugate comprising a second antibody or antigen-binding fragment. Signal generation is based on assessing the presence of a signal from a detectable label, and the presence of the signal indicates the presence of the N protein from the SARS-CoV-2 virus, or a fragment thereof, in the sample. The first and second binding reagents bind different epitopes on the target nucleocapsid protein.
The first antibody or antigen-binding fragment is defined by complementarity determining region amino acid sequences for a heavy chain variable region and a light chain variable region, including a heavy chain variable region comprising a CDR1 amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 amino acid sequence of SEQ ID NO: 4, a CDR2 amino acid sequence of SEQ ID NO: 5, and a CDR3 amino acid sequence of SEQ ID NO: 6. The second antibody or antigen-binding fragment is also defined by specific CDR amino acid sequences for heavy and light chain variable regions.
The invention further includes a lateral flow device comprising a first antibody or antigen-binding fragment that specifically binds the nucleocapsid (N) protein and a second antibody or antigen-binding fragment that specifically binds a protein from the SARS-CoV-2 virus, where the first and second antibodies bind different epitopes. In the device format, the specified antibody binding fragments are defined by the same CDR sequence identifiers for the heavy and light chain variable regions, enabling the device to be configured to detect SARS-CoV-2 nucleocapsid via a detectable label associated with the second antibody.
Claims Coverage
Two independent claims are identified. The claim set primarily covers an immunoassay format using two N-protein binding antibodies/antigen-binding fragments with different epitope binding and defined CDR sequences, and a lateral flow device configured with first and second antibodies/antigen-binding fragments with corresponding CDR sequence identifiers.
Immunoassay using two N-binding antibodies with defined CDR sequences
A method of detecting SARS-CoV-2 virus in a sample obtained from a subject, wherein the method is an immunoassay comprising contacting the sample with a first antibody or antigen-binding fragment that specifically binds to the nucleocapsid (N) protein from SARS-CoV-2 and has a heavy chain variable region with CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, and a light chain variable region with CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6; contacting the sample with a conjugate comprising a second antibody that specifically binds to N protein and binds a different epitope than the first antibody and includes a heavy chain variable region with CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 10, and CDR3 of SEQ ID NO: 11 and a light chain variable region with CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 13, and CDR3 of SEQ ID NO: 14; and assessing the presence of a signal from a detectable label where presence indicates the presence of the N protein or a fragment thereof.
Lateral flow device with first and second epitope-distinct N-binding antibodies with defined CDR sequences
A lateral flow device comprising a first antibody or antigen-binding fragment that specifically binds to the nucleocapsid (N) protein from SARS-CoV-2 and binds a different epitope than the first antibody or antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment comprises a heavy chain variable region with CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, and a light chain variable region with CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6; and a second antibody or antigen-binding fragment that specifically binds to a protein from SARS-CoV-2 and comprises a heavy chain variable region with CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 10, and CDR3 of SEQ ID NO: 11, and a light chain variable region with CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 13, and CDR3 of SEQ ID NO: 14.
Coverage is directed to detecting SARS-CoV-2 using immunoassays and lateral flow device formats that rely on two antibodies/antigen-binding fragments binding different epitopes of the nucleocapsid (N) protein, with each binding reagent constrained by specified heavy-chain and light-chain variable-region CDR amino acid sequences identified by SEQ ID NOs.
Stated Advantages
Maintained sensitivity for detection of nucleocapsid variants with sensitivity equivalent to wild type.
Improvements reported when using Fab fragments versus full-length antibodies.
Absence of hook effect up to high concentrations, as evaluated in performance testing.
Clinical evaluation reported with clinical sensitivity and specificity versus RT-PCR.
Testing indicates cross-reactivity and interference evaluation relative to other viruses and sample components, including SARS-CoV as an example.
Documented Applications
Detecting SARS-CoV-2 nucleocapsid (N) antigen in samples obtained from a subject, including nasal/nasopharyngeal swab, saliva, mucus, and blood components (blood/serum/plasma).
Use in immunoassay formats including enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFA).
Use in a lateral flow device and kit context configured with lateral flow strip components and sample handling components including extraction buffer and controls.
Detection of SARS-CoV-2 nucleocapsid variants with maintained sensitivity.
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