Synthesis and characterization of a functional benzoylecgonine – bovine serum albumin conjugate for quantification of a humanized monoclonal anti-cocaine antibody

Abstract

We have developed a humanized monoclonal anti-cocaine antibody (h2E2), as a potential solution for cocaine use disorder. A key milestone was the development of an assay to quantify this monoclonal antibody (mAb) in animal and human blood. Thus, we synthesized a novel benzoylecgonine-1,4-diaminobutane-BSA (BE-diab BSA) conjugate as an antigen for quantifying h2E2 using ELISA. We report here the method of synthesis of this conjugate, BE-diab BSA, and assessment of its binding to the h2E2 mAb using an ELISA and a fluorescence quenching assay. Compared with four commercial BE-BSA conjugates, BE-diab BSA demonstrated markedly stronger mAb binding in ELISA—three of the commercial conjugates showed less than 10 % relative binding. Fluorescence quenching assays confirmed this binding superiority, with the commercial conjugates showing minimal mAb interaction, while BE-diab BSA induced robust intrinsic fluorescence quenching. SDS-PAGE analyses identified structural differences consistent with binding results between our functional conjugate and the commercial preparations. This functional and reproducible in-house conjugation has been integrated into a GLP-validated ELISA, which is now in use for pharmacokinetic analyses and for qualifying antibody release lots for clinical deployment.