A double-sandwich ELISA for identification of monoclonal antibodies suitable for sandwich immunoassays

Authors: Stanker, Larry; Hnasko, Robert

Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: 8/12/2014
Publication Date: 9/1/2015
Citation: Stanker, L.H., Hnasko, R.M. 2015. A double-sandwich ELISA for identification of monoclonal antibodies suitable for sandwich immunoassays. Methods in Molecular Biology. Springer Science and Business Media, New York. 216 pp. 2015. (Book Chapter.

Interpretive Summary: Immunoassay (IA) represents a powerful diagnostic tool used in biology and medicine. Immunoassays depend on the availability of specific antibodies capable of detecting the target of interest (eg., a specific protein, bacteria, or virus). One of the most common IA configurations is a sandwich assay. This format used two antibody molecules, a capture antibody to trap the target and a second or detector antibody that binds to the target capture antibody complex. For this format to work, the capture antibody must capture the target out of solution. Unfortunately, many monoclonal antibodies isolated using traditional methods bind the target but will not or only poorly capture the target antigen in solution. Thus, they are not suitable for use in a sandwich IA. In this manuscript we describe a simple screening approach useful for identifying monoclonal antibodies capable of performing as a capture antibody in a sandwich IA. This approach accelerates identification of useful antibodies for development of diagnostic immunoassays.

Technical Abstract: The sandwich immunoassay (sIA) is an invaluable technique for concentrating, detecting, and quantifying target antigens. The two critical components required are a capture antibody and a detection antibody, each binding a different epitope on the target antigen. The specific antibodies incorporated into the test define most of the performance parameters of any subsequent immunoassay regardless of the assay format: traditional ELISA, lateral-flow immunoassay, various bead-based assays, antibody-based biosensors, or the reporting label. Here we describe an approach for identifying monoclonal antibodies (mAbs) suitable for use as capture antibodies and detector antibodies in a sELISA targeting bacterial protein toxins. The approach was designed for early identification of monoclonal antibodies (mAbs), in the initial hybridoma screen.