Measurement of AgAb interaction energies

There is a large variety of equilibrium methods by which the parameters on the right-hand side of eqn (4) can be determined, allowing the calculation of Ka and from this value, of K'., and of AG. Among these methods are equilibrium dialysis or ultrafiltration, precipitation methods, analytical ultracentrifugation, gel filtration or size exclusion chromatography, affinity chromatography, fluorescence, fluorescence quenching, fluorescence polarization, immunoassay methods, hapten inhibition methods, nuclear magnetic resonance, electron spin resonance, etc. The powerful new bioaffinity sensor methods should be specially mentioned.

Bioaffinity sensors

The bioaffinity sensor methodology has, since the early 1990s, developed into one of the most powerful approaches to determining the kinetic association (ka) and dissociation (kd) constants, and from these the equilibrium binding constant, Ka (see eqn (3), above), and its inverse, the equilibrium dissociation constant, Kd = kjk^. By optical means, the thickening of a layer of, for example, Ab, which is continuously deposited by means of a thin stream of an Ab solution moving across a thin layer of Ag molecules, can be monitored in real time. The graph of Ab thickness versus time thus obtained yields the kinetic association constant, ka. Upon subsequent replacement of the Ab solution with a buffer solution, the kinetic dissociation constant, kd, is obtained. From these the equilibrium binding constant, K„ is derived (see eqn (3)).

Concentration dependence of Ka

With polyclonal Abs, for example, in the BSA-anti-BSA system (BSA = bovine serum albumin), 100-fold dilution (at Ag/Ab equivalence) causes an almost 40 000 fold increase in iCa, i.e. from 1.7 xlO7 to

6.3 x 1011 1 mol-1. Even with monoclonal Abs a significant increase in K, with dilution must be expected because greater dilution minimizes steric hindrance. Thus, if one aims to characterize an Ag-Ab system by measuring the iCa value, it is advisable to operate at the lowest practical reagent concentration. For instance in a Biosensor setting, with a constant Ag concentration in the immobilized phase, a 100-fold decrease in the mobile phase (monoclonal Ab solution) typically gives rise to a 70- to 100-fold decrease in ka and iCa. In general, under similar circumstances, only the lowest feasible Ab concentration in the mobile phase will yield accurate and Ka values.

See also: Affinity; Antigens; Immunoglobulin structure; Precipitation reaction; Surface plasmon resonance; Valency of antigens and antibodies.

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