Crosslinking of Antibody Molecules by Bifunctional Antigens

Abstract

A requirement for at least two antigenic determinants to induce humoral antibody responses has been demonstrated using derivatives of a small molecule, L-tyrosine-p-azobenzenearsonate (RAT). This molecule itself induces only cellular immunity in guinea pigs. Assymetric bifunctional antigens composed of one RAT moiety and one haptenic determinant, such as DNP, with or without a spacer, induce cellular immunity to RAT and anti-DNP antibody. A symmetrical bifunctional antigen comprised of two RAT determinants separated by a rigid spacer, (PRO)₁₀, induces cellular and humoral responses, but the same two functions separated by a flexible spacer (6-amino-caproyl) gives cellular responses only. The bifunctionality of the latter antigen is probably compromised by intramolecular stacking of azoarsonate groups, since this molecule exhibits extensive hypochromism in physiological solution. The simplest hypothetical model of cell cooperation leading to a humoral response, i.e., bridging of T and B cells by antigen, requires the congregation of two lymphocytes on a molecule as small as DNP-RAT. Since the means of direct demonstration of such an interaction are not available, the capacity of such bifunctional molecules to bridge the receptors of specific antibody molecules was examined here. Thin layer gel chromatography was used to assess the crosslinking of the specific antibodies. It was found that all the bifunctional molecules examined were able to polymerize specific antibodies, regardless of whether they were effective mediators of cell cooperation in vivo.