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.
Highlights
Β-Lactam antibiotics, penicillins, are broadly used in veterinary practice
The Amp-pPh-horseradish peroxidase (HRP) enzyme conjugate was used in direct enzyme-linked immunosorbent assay (ELISA) at 0.3 μg/mL; the Amp-Ad-HRP and Amox-pPh-HRP conjugates were used at 0.5 μg/mL
New activated esters of carboxy aminopenicillin derivatives were obtained via acylation of Amp and Amox with di-OSu esters of dicarboxylic acids
Summary
Β-Lactam antibiotics, penicillins, are broadly used in veterinary practice. Immunochemical methods provide an alternative, being rapid and relatively inexpensive Their development and application requires protein reagents (receptors and antibodies) that are capable of highly sensitive penicillin binding, as well as high-molecular-weight derivatives of the antibiotics as haptens: enzyme conjugates, immunogens, and conjugated antigens. The group includes a high-molecular-weight membrane transpeptidase PBP2x of Streptococcus pneumoniae R6 [7] and the carboxy terminal domain of the β-lactam transmembrane receptor (BlaR-CTD) of Bacillus licheniformis [8]. These proteins recognize the antibiotic structure that contains the β-lactam cycle and bind various β-lactams, including both penicillins and cephalosporins. Immunochromatographic assays for a broad range of β-lactams have been performed using PBP [18, 19] and an Amp-specific monoclonal antibody [20]
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