Characterization and optimization of heroin hapten-BSA conjugates: method development for the synthesis of reproducible hapten-based vaccines

A potential new treatment for drug addiction is immunization with vaccines that induce antibodies that can abrogate the addictive effects of the drug of abuse. One of the challenges in the development of a vaccine against drugs of abuse is the availability of an optimum procedure that gives reproducible and high yielding hapten-protein conjugates. In this study, a heroin/morphine surrogate hapten (MorHap) was coupled to bovine serum albumin (BSA) using maleimide-thiol chemistry. MorHap-BSA conjugates with 3, 5, 10, 15, 22, 28, and 34 haptens were obtained using different linker and hapten ratios. Using this optimized procedure, MorHap-BSA conjugates were synthesized with highly reproducible results and in high yields. The number of haptens attached to BSA was compared by 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, modified Ellman's test and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Among the three methods, MALDI-TOF MS discriminated subtle differences in hapten density. The effect of hapten density on enzyme-linked immunosorbent assay (ELISA) performance was evaluated with seven MorHap-BSA conjugates of varying hapten densities, which were used as coating antigens. The highest antibody binding was obtained with MorHap-BSA conjugates containing 3-5 haptens. This is the first report that rigorously analyzes, optimizes and characterizes the conjugation of haptens to proteins that can be used for vaccines against drugs of abuse. The effect of hapten density on the ELISA detection of antibodies against haptens demonstrates the importance of careful characterization of the hapten density by the analytical techniques described.