Phylogeny of immunoglobulin structure and function. V. Valences and association constants of teleost antibodies to a haptenic determinant.

The giant grouper, Epinephelus itaira, was shown to synthesize 16 and 6.4S antibodies specific for the dinitrophenyl determinant (DNP). Sera obtained at various intervals between 1 month and 2 yr after initial immunization contained both species of antibody; no temporal synthesis was evident. Equilibrium dialysis studies employing epsilon-dinitrophenyl-amino caproic acid were conducted with purified grouper antibodies specific for DNP. The 16S antibody preparations obtained at 1 and 2 months of immunization showed heterogeneity of hapten binding indicative of two populations of combining sites. One-half of these sites (an average of four sites per 16S molecule) exhibited an average intrinsic association constant (K(o)) of approximately 10(6)M(-1); the K(o) of the remaining four-sites was approximately 10(4)M(-1). Thus, the valence of the grouper 16S antibody molecule appears to be eight although the distribution of the high and low K(o) sites is unknown, i.e., are they each on the same or on different molecules? The 16S antibody preparations obtained after more prolonged immunization exhibited increasingly lower K(o) values; the so-called low K(o) sites were no longer detectable. These findings are in contrast to reports of rabbit IgG antibodies showing an increase in K(o) with increased time. The 6.4S antibody preparations obtained from the 1 and 2 month antisera had K(o) values of approximately 10(6)M(-1) and a valence of one. These antibodies would not precipitate with antigen. The 6.4S antibody preparations obtained at later times showed decreasing K(o) values comparable to those of the 16S antibodies from the same bleedings. Studies on the thermodynamic parameters of the hapten-antibody interaction showed the grouper 16 and 6.4S antibodies to be similar to each other. These data also showed that the enthalpy and entropy changes of grouper antibody-hapten reactions resemble those reported for rabbit IgG antibodies to this hapten. It is thus suggested that, although considerable evolution of immunoglobulin classes has occurred between fish and rabbits, the antibody combining site may have remained relatively unchanged during a large part of evolutionary time.