Restricted reassociation of heavy and light chains from hapten-specific monoclonal antibodies.

AUTOR(ES)

Kranz, D M

RESUMO

Six murine monoclonal antifluorescyl antibody clones encompassing a defined range of affinities and containing kappa light chains with IgG1 or IgG2 heavy chains were examined. As the fluorescence of the ligand is quenched greater than 90% when fluorescein is bound by antifluorescyl antibodies, fluorescence quenching was assayed to monitor polypeptide reconstitution and active site formation on mixing of resolved heavy (H) and light (L) chains. Of 36 possible experimental combinations of H- and L-chain reaction mixtures, only homologous H and L chains (derived from the same parental immunoglobulin molecule) bound fluorescein. Results from fluorescence polarization studies, conducted independently of fluorescence quenching, confirmed the findings. Competitive inhibition and molecular sieve experiments showed that, despite preferential association of homologous H and L chains, several heterologous H and L chains associated to form intact 7S molecules, although no active site was constituted. Thus, polypeptide recombination and formation of functional antigen binding sites are two processes that immunocytes must regulate during cell differentiation and generation of diversity. A mechanism and underlying the observed preferential reassociation of specific H and L chains and a means of generating affinity maturation, as exhibited by the antifluorescein system, is proposed.

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