Binding kinetics of monoclonal antibody using antigen-β-galactosidase hybrid protein: application to measurement of peptide antigenicity

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Title
Binding kinetics of monoclonal antibody using antigen-β-galactosidase hybrid protein: application to measurement of peptide antigenicity
Author(s)
Eun Wie Cho; Myung Kyu Lee; Kil Lyong Kim; Kyung Soo Hahm
Bibliographic Citation
Journal of Immunoassay and Immunochemistry, vol. 16, no. 4, pp. 349-363
Publication Year
1995
Abstract
A simple method for determination of binding kinetics of a solid-phase antibody using antigen-β-galactosidase hybrid protein was evaluated. To minimize conformational change of the antigen binding site of the antibody when directly binding to a microtiter plate, the microtiter plate was precoated with protein A. The binding and free antigen concentrations were directly obtained from the β-galactosidase activity. This method can be used for analyses of the equilibrium dissociation constant (K(D)), and the association (K(ass)) and dissociation (Kd(iss)) rate constants. Peptide antigenicity was also analyzed by competitive ELISA using this method. Since both antigen-β-galactosidase and the peptide used are localized in the fluid-phase, the proper affinity constant (K(A)) of the peptide can be estimated from the K(D) value of the antigen-β-galactosidase-antibody interaction, and from the IC50 value of the peptide.
Keyword
antigen-β-galactosidase hybrid proteinBinding kineticscompetitive ELISAmonoclonal antibodypeptide
ISSN
0197-1522
Publisher
Taylor & Francis
Type
Article
Appears in Collections:
Division of Biomedical Research > Rare Disease Research Center > 1. Journal Articles
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