Effect of Interaction between Hapten-Specific Cells Preselected for Different Receptor Affinities and Carrier-Primed Cells on Antibody Avidity

Abstract

Our previous studies,in vivo (1) and in vitro (2), have shown that carrier priming enhances antibody response and avidity. Under optimal conditions for these carrier effects to occur, C3HeB/FeJ and (C57BI/10×DBA/2)F1 mice either carrier-primed with HRBC or -unprimed were given the conjugate TNP-HRBC. Thereafter, spleen cells from the carrier-primed or -unprimed mice were daily pooled and assayed by the hemolytic plaque technique with TNP-SRBC to determine the number of direct PFC anti-TNP. Avidity of antibodies secreted by PFC was estimated from inhibition of PFC by soluble TNP-BSA (35 mols TNP/mol BSA). Aliquots of TNP-BSA dissolved in PBS at different concentrations and aliquots of spleen cells were plated in agar containing TNP-SRBC. The number of direct PFC developed after addition of guinea pig complement was decreased by TNP-BSA. Percent inhibitions of PFC were transformed to probits which appeared linearly related to the log dose of inhibitor. The reciprocal of the dose of inhibitor that suppressed 50% of the PFC was taken as an estimate of avidity: the higher the antibody avidity, the lower the inhibitor dose and the higher its reciprocal. The dose of inhibitor was referred to TNP-lysyl residues and calculated from spectrophotometric measurements of the TNP-BSA solution at λ max 348 nm by assuming 15,400 as the molar extinction coefficient for the TNP-lysyl residue. In each PFC inhibition assay, spleen cells were exposed to amounts of TNP-lysyl residues ranging from 10⁻⁵ to 10⁻¹ mg.

Supported by CNEN-Euratom Association Contract. Publication No. 1209 of the Euratom Biology Division