Homopolyvalent antibody-antigen interaction kinetic studies with use of a dual-polarization interferometric biosensor.
We used dual-polarization interferometry (DPI) to study the interaction kinetics between a 'homopolyvalent' antigen (Ag) and a monoclonal antibody (Ab). A model system, which uses a monoclonal Ab against a homopentameric Ag, C-reactive protein (CRP), is presented with principle and experiments for the study of the interactions between an Ab and an Ag that has multiple identical epitopes. This allows evaluation of the dissociation constant (K(D)) and of the binding stoichiometry by DPI based on measurements of phase changes of Ab-Ag complexes in the transverse magnetic (TM) and transverse electric (TE) polarization modes. The average experimental value of K(D) found by the DPI technique for anti-CRP Ab was shown to be in close agreement with the value obtained by an indirect competition-enzyme-linked immunosorbent assay (ELISA). Moreover, the total number of Ab combining sites on the DPI sensor chip was calculated, and the binding stoichiometry of the surface Ag-Ab complex was obtained. This study illustrates the advantages of the DPI method in biosensing in its capacity for simultaneous evaluation of the thickness and refractive index (density, mass) of adsorbed layers. This allowed a comprehensive analysis of affinity reactions between an Ab having two binding sites and a multi-sited Ag.
Lin
Shiming
S
Center for Optoelectronic Biomedicine, National Taiwan University, 1-1 Jen-Ai Road, Taipei, Taiwan. sml@ntumc.org
Lee
Chih-Kung
CK
Lin
Yin-Hang
YH
Lee
Shih-Yuan
SY
Sheu
Bor-Ching
BC
Tsai
Jui-Chang
JC
Hsu
Su-Ming
SM
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000032:analysis* / Q000276:immunology*; D000937:Antigen-Antibody Reactions / Q000276:immunology; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002097:C-Reactive Protein / Q000276:immunology*; D003198:Computer Simulation; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D007700:Kinetics; D008857:Microscopy, Interference / Q000295:instrumentation / Q000379:methods; D008859:Microscopy, Polarization / Q000295:instrumentation / Q000379:methods; D018448:Models, Immunological
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002097:C-Reactive Protein