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. 2024:2768:15-27.
doi: 10.1007/978-1-0716-3690-9_2.

Current Trends in Validating Antibody Specificities for ELISpot by Western Blotting

Biji T Kurien  1   2 , R Hal Scofield  3   4   5
Affiliations

Current Trends in Validating Antibody Specificities for ELISpot by Western Blotting

Biji T Kurien et al. Methods Mol Biol. 2024.

Abstract

The enzyme-linked immunospot (ELISpot) assay is a highly useful and sensitive method to detect total immunoglobulin and antigen-specific antibody-secreting cells. In addition, this method can measure biological activity and immunological secretions from immune cells. In general, membrane-bound antigen allows binding of antibody secreted by B cells, or a membrane-bound analyte-specific antibody binds to the specific analyte (e.g., cytokines) elicited from cells added to the well containing the bound antibody. The response from added cells is then detected by using an anti-Ig antibody and a colorimetric substrate, while in the case of non-B cells, the elicited antigen is detected with appropriate antibodies and enzyme-conjugated antibodies. Specificity of antibodies binding the protein of interest is necessary to achieve correct results. Western blotting can be used for this with/without siRNA knockdown of proteins of interest or with the use of peptide inhibitors to inhibit the binding of specific antibodies to the target protein. Despite its general simplicity, western blotting is a powerful technique for immunodetection of proteins (notably low abundance proteins) as it provides simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Now, we have plethora of immunoblotting methods to validate antibodies for ELISpot.

Keywords: Nitrocellulose; SDS-PAGE; Western blotting.

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