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Chain Interactions and Idiotypic Specificities of Homogeneous Rabbit Anti-Lactose Antibodies¹

From the Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174

Abstract

Functionally homogeneous fractions of rabbit anti-lactose IgG antibody have been characterized with respect to their idiotypic specificity and the effect of homologous recombination of isolated chains on the recovery of functional homogeneity and idiotypic determinants. Dimeric heavy chains and monomeric light chains were obtained from homogeneous antibody fractions and used for the preparation of reconstituted IgG molecules in high yield. The isolated chains were also evaluated with respect to binding affinity and inhibition of anti-idiotype antibody. The most significant observation was the recovery of idiotypic specificity in the homologous recombinants as well as binding affinity and functional homogeneity. These results provide strong evidence that the homogeneous antibody fractions are monoclonal products.

The isolated chains exhibited little or no specific activity. With respect to binding, the dimeric heavy chain bound a lactosyl-containing ligand with an association constant about 100-fold less than that of the parent antibody. Although the binding was homogeneous only one binding site per dimer was expressed. The heavy chain was also found to inhibit slightly the anti-idiotype reaction. The isolated monomeric light chain, on the other hand, showed no reactivity in either kind of experiment. The anti-idiotype reaction was subject to inhibition with lactose and p-amino-phenyl--lactoside with some antisera, depending on the form in which the homogeneous antibody was presented for immunization. It is suggested that the major portion of the idiotypic determinant is constituted of amino acid residues located along the periphery of the binding site rather than the contact residues in the groove or cleft of the site. This proposal would account for the inhibition by ligands as the result of the inability of ligand and anti-idiotype antibody to be accommodated simultaneously at contiguous sites because of overlapping volumes. Those cases in which ligand inhibition is not observed could be attributed to unusual geometric relationships between the contiguous sites.

Footnotes

¹ This work was supported in part by Public Health Service Research Grant AI-09492 from the National Institute of Allergy and Infectious Diseases and in part by Public Health Service Training Grant 5-TI-AI-204 from the National Institute of Allergy and Infectious Diseases.

² Present address: W.H.O. Immunoglobulin Laboratories, 21 rue du Bugnon, 1005 Lausanne, Switzerland.

³ Recipient of Public Health Service Research Career Award 5-K6-AI-14,012 from the National Institute of Allergy and Infectious Diseases.