Conjugates of Aminopenicillins with Proteins: Synthesis, Immunogenic Properties, and Binding to the β-Lactam Receptor and Antibodies

Abstract

A new approach to aminopenicillin modification and conjugation with proteins was developed using di-N-hydroxysuccinimide esters of dicarboxylic acids as crosslinkers. Acylation of ampicillin (Amp) and amoxicillin (Amox) with di-N-hydroxysuccinimide esters of adipic or terephthalic acids was carried out in an organic solvent. Subsequent conjugation of the resulting aminopenicillin derivatives with proteins was done in an aqueous medium at pH 8.3 to produce immunogenic and enzymatic conjugates of Amp and Amox. The β-lactam cycle of Amp was shown to remain intact after chemical modification and synthesis of linker conjugates. An immunogenic Amp–thyroglobulin conjugate containing an aromatic linker was used for long-term immunization of rabbits, and polyclonal antibodies thus obtained were found to bind Amp, Amox, and penicillin G with extremely high sensitivity. Amp and Amox conjugates with horseradish peroxidase (HRP) were synthesized and characterized in a competitive protein-binding (receptor) assay and a direct competitive enzyme-linked immunosorbent assay (ELISA). Of the model immunoassay systems tested, the best characteristics were observed for heterologous direct ELISA with polyclonal antibodies and the Amp–HRP conjugate that contained an adipic acid fragment as a linker: the Amp sensitivity was 0.03 ng/mL and IC50 = 0.20 ng/mL.