Use of a resonant mirror biosensor to characterize the interaction of carboxypeptidase A with an elicited monoclonal antibody.
The binding of apocarboxypeptidase A to an immobilized form of its elicited monoclonal antibody has been used to explore the potential of a biosensor instrument (IAsys) based on resonant mirror technology for the quantitative characterization of antibody-antigen interactions. Advantage has been taken of the stirred cuvette design of the IAsys instrument to develop a stepwise titration procedure for thermodynamic characterization of the interaction, an association equilibrium constant of 3.3 (+/-0.9) x 10(7) M-1 having been obtained under the conditions studied (0.1 M Tris/HCl-0.5 M NaCl, pH 7.5, 21 degrees C). In a test of the feasibility of subjecting the time course of biosensor response to conventional pseudo-first-order kinetic analysis, nonconformity of results with such description was encountered at high and low concentrations of apocarboxypeptidase A. Whereas the deviations from Langmuirian kinetic behavior at high antigen concentrations undoubtedly stem from the same sources as those already encountered in studies with the BIA-core biosensor instrument, the deviations at the other concentration extreme occur in a range in which the assumed constancy of free antigen concentration in the liquid phase is becoming a poor approximation. An alternative approach in such circumstances has been tested in which prior thermodynamic characterization is a prerequisite for rate constant evaluation by means of a second-order kinetic analysis. Finally, the effect of soluble anticarboxypeptidase A on the pseudo-first-order kinetics of the biosensor response has been used to illustrate a simple kinetic procedure for evaluating the affinity constant for the antibody-antigen interaction in solution, a value of 1.9 (+/-0.2) x 10(8) M-1 being obtained by such means.
Hall
D R
DR
Department of Biochemistry, University of Queensland, Brisbane, Australia.
Winzor
D J
DJ
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D015374:Biosensing Techniques; D002268:Carboxypeptidases / Q000276:immunology*; D043422:Carboxypeptidases A; D007700:Kinetics; D017186:Titrimetry
D000911:Antibodies, Monoclonal; D002268:Carboxypeptidases; D043422:Carboxypeptidases A