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Title 

A convenient method for epitope competition analysis of two monoclonal antibodies for their antigen binding

Authors 

Ju Won KwakChang-Soon Yoon

Publisher 

Elsevier

Issue Date 

1996

Citation 

Journal of Immunological Methods, vol. 191, no. 1, pp. 49-54

Keywords 

anti-apolipoprotein antibodyantigen-binding sitedetecting antibodyELISA, sandwichHRP couplingimmunoassayapolipoproteinepitopemonoclonal antibodyenzyme-linked immunosorbent assay

Abstract 

Choosing optimum pair of capturing antibody and detecting antibody when developing monoclonal antibody (MAb)-based, sandwich enzyme-linked immunosorbent assays is a time-consuming process requiring the coupling of individual antibodies to an enzyme like horseradish peroxidase or alkaline phosphatase. The MAbs required for the two-site sandwich ELISA should bind to distinct epitopes of the antigen, and their binding should not be mutually exclusive. To determine if two monoclonal antibodies would occupy distinct sites of their antigen in binding, an enzyme-linked immunosorbent assay was devised, which is easy-to-use and does not require any coupling of monoclonal antibodies to enzymes. Microplate wells are coated with rabbit polyclonal antibodies raised against the same antigen of MAbs. After blocking, a limited amount of the antigen is added for incubation with the rabbit antibodies. Mouse monoclonal antibody 1 (MAb 1) is added to saturation. A serial dilution of MAb 2 (for analysis) or MAb 1 (for control) is added subsequently. An enzyme-labeled, goat anti-mouse secondary antibody and its substrates are added for color development. Thus, the epitope competition of two MAbs for their antigen binding is easily determined by the measurement and comparison of color development between the two MAb additions.

ISSN 

0022-1759

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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