Coupling of large haptens to proteins and cell surfaces: Preparation of stable, optimally sensitized erhythrocytes for hapten-specific, hemolytic plaque assays

Abstract

Procedures are described for converting tertiary-butyloxycarbonyl (Boc) hydrazides of large haptenic structures to reactive acyl azides and for using these intermediates in the preparation of a sereis of highly specific immunochemical reagents. The synthesis of the initial C-terminal Boc hydrazides, which are tripeptide derivatives, is described in a companion report (Inman et al. (1973) Immunochemistry 10, 153). The converted azides react readily and specifically with amino groups in mildly alkaline, aqueous media and thereby provide an excellent means for covalently coupling haptens to immunogen carriers, proteins, or cells. The azide groups have relatively uniform reactivity due to an adjacent, common, glycylglycyl structure. Thus, it has been possible to prepare groups of immunochemical reagents, representing numerous specificities, by use of a single convenient procedure. Erythrocytes bound with large haptens by this method have functioned as highly stable and sensitive indicator cells in hapten-specific hemolytic plaque and hemagglutination assays. Conditions for preparing optimally sensitized erythrocytes for quantitative studies of hapten-specific plaque forming cells have been studies. Maximum numbers of plaque forming cells were detected when about 300,000 hapten molecules were bound per indicator erythrocyte. Plaque counts decreased progressively when indicator red cells had been conjugated with appreciably more or less than this amount of hapten. It has not been previously recognized that excessively hapten derivatization of erythrocytes regularly and profoundly impedes their susceptibility to specific immune lysis. Estimates have been made of the molecular circumstances which may explain this phenomenon. Its generaly implications for complement mediated cytolysis are discussed.