The potential of monoclonal antibodies against ampicillin for the preparation of a multi-immunoaffinity chromatography for penicillins†

Abstract

Monoclonal antibodies (Mab) against ampicillin were prepared by immunization of mice with an ampicillin–keyhole limpet hemocyanin conjugate coupled by a glutaraldehyde method. Sensitivity and specificity of these antibodies were tested in a direct competitive enzyme immunoassay, in which an ampicillin–horseradish peroxidase conjugate prepared by a carbodiimide method served as the labelled antigen. According to their cross-reactivities with the other β-lactam antibiotics, the Mabs could be divided into two groups, which are represented by the clones designated 1D1 and 3B5. While Mab 3B5 (IgG₁) showed no major cross-reactions with the other penicillins frequently used in veterinary medicine except for amoxicillin (108%), Mab 1D1 (IgG_2a) had marked cross-reactivities with most of the 17 tested β-lactam antibiotics (e.g., amoxicillin 187%, penicillin G 31%, cloxacillin 30%, dicloxacillin 44%, and oxacillin 14%). The detection limits for ampicillin, calculated from the antibiotic concentration giving 30% binding inhibition, were 11.7 (Mab 3B5) and 16.6 ng ml^–1 (Mab 1D1). To prepare multi-immunoaffinity chromatography columns, Mab 1D1 and a previously described antibody against cloxacillin (Mab 1F7) were each coupled to CNBr activated sepharose. The capacity of the resulting immunosorbents was approximately 6.6 and 5.4 µg ml^–1 gel for ampicillin and cloxacillin, respectively. Recoveries of amoxicillin, ampicillin, cloxacillin, dicloxacillin, penicillin G and oxacillin (in buffer solutions) from the produced immunoaffinity columns were in the range from 67 to 100%.