MEASUREMENT OF AFFINITY IN SERUM SAMPLES OF ANTIGEN-FREE, GERM-FREE AND CONVENTIONAL MICE AFTER HYPERIMMUNIZATION WITH 2,4-DINITROPHENYL KEYHOLE LIMPET HEMOCYANIN, USING SURFACE-PLASMON RESONANCE

We previously investigated the primary and secondary responses and hyp erimmunization to the T cell-dependent antigen 2,4-dinitrophenyl keyho le limpet hemocyanin (DNP-KLH) in antigen-free (AF), germ-free (GF) an d conventional (CV) mice. Both the absolute and relative numbers of DN P-specific IgG-secreting cells in the spleen of AF mice were considera bly higher compared to GF and CV mice, especially after hyperimmunizat ion. In the present study we measured the total and DNP-specific IgG c oncentration in the sera of these hyperimmunized mice using a sensitiv e sandwich enzyme-linked immunosorbent assay. With respect to the tota l IgG concentration before and after hyperimmunization, the AF mice sh owed an almost 13-fold increase after boosting with the antigen; the G F mice showed an approximately 8-fold increase. A slight but non-signi ficant increase was observed in the CV mice. The total as well as the DNP-specific IgG levels in the AF-immunized mice were 2-fold and 5-fol d higher compared to GF and CV mice, respectively. With the use of Sur face Plasmon Resonance instrumentation (BIAcore(TM), Pharmacia, Uppsal a, Sweden) we obtained mean binding affinities (K-A) of the polyclonal samples of the three groups of hyperimmunized mice. IgA and IgM sampl es displayed low affinity for DNP-lysine. The AF mice displayed the hi ghest K-A value among IgG antibodies, followed by GF mice, while CV mi ce showed a 3-fold lower K-A compared to AF mice. These differences we re mainly determined by the dissociation rate constant (k(diss)), sinc e no significant changes were observed in the association rate constan t (k(ass)). Furthermore, the sera of the CV mice have a lower percenta ge of high-affinity antibodies compared to GF and AF mice. These resul ts suggest that besides a higher overall binding affinity seen in AF m ice, and to a lesser extent in GF mice, the relative contribution of h igh-affinity IgG is greater in AF mice compared to CV mice.