Abstract— Fifteen protein conjugates of penicillins and cephalosporins containing amino- and/or carboxylic groups in the initial structures have been synthesized in the reactions with human serum albumin or ovalbumin using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) or a combination of EDC and N-hydroxysulfosuccinimide at various ratios of the base reagents. A comparative study of conjugates composition and properties has been carried out by UV spectroscopy, mass spectrometry and a ligand-receptor assay. It was shown that the antibiotic residue content of the macromolecules obtained varied from 1 to 22, the beta-lactam cycle remained intact assuring specific interactions of the conjugates with a penicillin-binding protein. In two developed models of receptor bioanalytic systems, an ampicillin conjugate onto a solid phase binds to penicillin-binding protein complexed with a monoclonal antibody, which was detected by an immunoenzyme label in microplate wells or gold nanoparticles on test strips. Conjugated ampicillin binding to the receptor was competitively inhibited by beta-lactam antibiotics added to the liquid phase, and analytical sensitivities relative to penicillin G were 0.05 and 1 ng/mL for microplate and receptor chromatographic systems, respectively.

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.

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