Chemical basis for diversity in antibody specificity analysed by hapten binding to monoclonal anti-4-hydroxy-3-nitrophenacetyl (NP) immunoglobulins

Anti-4-hydroxy-3-nitrophenacetyl (NP)2 monoclonal antibodies were produced by hybrid cell lines obtained by fusion of NSl myeloma cells with lymphocytes from spleens of C57BL/6 mice immunized with NP conjugated with chicken immunoglobulin (NP-CG). Equilibrium constants of four purified immunoglobulins for N¹²⁵IP-ε-aminocaproate, measured by equilibrium dialysis at 4°C, ranged from 1.0 × 10^?8 to 1.0 × 10^?7M. In order to probe fine-specificity differences, binding constants were measured for a group of structural analogs of NP. This study showed that all four antibodies were heteroclitic and bound the iodo group of NIP-ε-aminocaproate with similar affinities, while the affinity constants for the ε-aminocaproate moiety varied widely. The free energies of binding for iodo group and the side-chain moieties were additive, whereas those for the hydroxy and nitro groups were not. Three of the immunoglobulins bound the aromatic ring system of NIP-ε-aminocaproate similarly, but less effectively than the fourth antibody. The data suggest that anti-NP active sites of diverse specificities were generated on the basis of additive interactions with multiple subsites.