AN IMPROVED METHOD FOR THE PRODUCTION OF ANTIBODIES TO LIPOPHILIC CARBOXYLIC HAPTEN USING SMALL AMOUNT OF HAPTEN-CARRIER CONJUGATE

A rapid, simple and low cost procedure for preparing minute amount of hapten-protein conjugates was developed using 4-acetyl benzoic acid (A BA) and two other closely related small chromophoric haptens. The amid e bond-generating mixed anhydride method of Erlanger was modified to p romote conjugation to various proteins (bovine serum albumin, ovalbumi n, casein and hemocyanin) or to a synthetic homopolymer (Poly-DL-lysin e). The key process in this synthesis is the use of a reversed micella r medium allowing strong carrier haptenization as determined by spectr ophotometric measurement at characteristic hapten absorption peaks. Th is coupling procedure is applicable to as little hapten material as 0. 2 mu mol and is disclosed to be most valuable for other rare lipid hap tens which pose analytical problem in biological fluids and matrices. Specific mice polyclonal antibodies were produced following multiple i ntraperitoneal injections of (ABA)(23)-BSA conjugate as revealed by in direct and competitive ELISA. Calculated K-D for the interaction of th e antibodies with free ABA was found to be 5 x 10(-5) M. (C) 1998 Publ ished by Elsevier Science B.V. All rights :reserved.