Analysis of heterogeneous dissociation kinetics in polyclonal populations of rabbit anti-fluorescyl-IgG antibodies.

Hapten dissociation kinetics were used to characterize the expression of hyperimmune anti-fluorescyl-IgG antibodies within a population of 14 New Zealand White (NZW) rabbits. Anti-fluorescyl-IgG antibody preparations, obtained from individual hyperimmune NZW rabbits, exhibited either three or four discrete dissociative components. The fastest component was typically heterogeneous with respect to dissociation rate, exhibiting an average dissociative lifetime of 8 sec, where dissociative lifetime was the reciprocal dissociation rate constant. Longer-lifetime components exhibited lifetimes of several hundred seconds, and became progressively more homogeneous with increasing lifetime. Comparison of dissociative components, within the NZW rabbit population, revealed the presence of four distinct groupings of dissociative lifetimes, which were approximately Gaussian in their distribution. Average lifetimes of 8, 52.6, 313 and 1417 sec were observed for the four groupings, which corresponded to free-energy (delta G0) values of -9.6, -10.6, -11.6 and -12.42 kcal/mole, respectively (using an association rate value of 5 X 10(6) M-1 sec-1). These findings implied that affinity maturation of the anti-fluorescyl-IgG antibody response involved the generation of discrete high-affinity subpopulations of antibodies rather than a continuous spectrum of high-affinity antibodies.