Selective Binding of Polychlorinated Biphenyl Congeners by a Monoclonal Antibody:  Analysis by Kinetic Exclusion Fluorescence Immunoassay

• Published in: Analytical chemistry (Washington) Vol. 73; no. 22; pp. 5477 - 5484
• Main Authors: Chiu, Ya-Wen, Li, Qing X, Karu, Alexander E
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15-11-2001
• Subjects: Antibodies, Monoclonal - immunology; Antibody Specificity; Antigen-Antibody Reactions; Biological and medical sciences; Chemistry; Fluorescence; Fundamental and applied biological sciences. Psychology; Immunoassay - methods; Immunoassay - standards; Kinetics; Molecular and cellular biology; PCB; Polychlorinated biphenyls; Polychlorinated Biphenyls - analysis; Polychlorinated Biphenyls - immunology; Sensitivity and Specificity; Spectrometry, Fluorescence
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac0102462

A previously described monoclonal antibody, S2B1, was highly selective for coplanar (non-ortho-chlorinated) PCB congeners in enzyme immunoassays that measured binding at equilibrium. In the present study, kinetic exclusion fluoroimmunoassay (KinExA) was used to determine the dissociation constants (K d) and on and off rates (k on, k off ) for binding of various PCB congeners to affinity-purified S2B1 IgG and Fab fragments in solution. This method revealed that mono- and di-ortho-chlorinated PCBs were bound by S2B1, but the on rates were slower, and the off rates faster by 6−60-fold, than with congeners that had no ortho chlorines. Although the sensitivity of immunoassays may be improved by using competing haptens that S2B1 binds more weakly than the parent PCB, the KinExA results demonstrate that congener specificity is an intrinsic property of S2B1 and does not require weaker binding haptens. KinExA also provided new information on the percentage of active binding sites, valence, and effects of buffer, solvent, and biotinylation on S2B1. The advantages and drawbacks of KinExA for measuring antibody−ligand binding are described.