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

Monoclonal antibodies (Mab) against ampicillin were prepared by immunizatio n of mice with an ampicillin-keyhole limpet hemocyanin conjugate coupled by a glutaraldehyde method. Sensitivity and specificity of these antibodies w ere tested in a direct competitive enzyme immunoassay, in which an ampicill in-horseradish peroxidase conjugate prepared by a carbodiimide method serve d as the labelled antigen. According to their cross-reactivities with the o ther beta-lactam antibiotics, the Mabs could be divided into two groups, wh ich are represented by the clones designated 1D1 and 3B5. While Mab 3B5 (Ig G(1)) showed no major cross-reactions with the ether penicillins frequently used in veterinary medicine except for amoxicillin (108%), Mab 1D1 (1gG(2a )) had marked cross-reactivities with most of the 17 tested beta-lactam ant ibiotics (e.g., amoxicillin 187%, penicillin G 31%, cloxacillin 30%, diclox acillin 44%, and oxacillin 14%). The detection limits for ampicillin, calcu lated from the antibiotic concentration giving 30% binding inhibition, were 11.7 (Mab 3B5) and 16.6 ng ml(-1) (Mab 1D1). To prepare multi-immunoaffini ty chromatography columns, Mab 1D1 and a previously described antibody agai nst cloxacillin (Mab 1F7) were each coupled to CNBr activated sepharose. Th e capacity of the resulting immunosorbents was approximately 6.6 and 5.4 mu g ml(-1) gel for ampicillin and cloxacillin, respectively. Recoveries of a moxicillin, ampicillin, cloxacillin, dicloxacillin, penicillin G and oxacil lin (in buffer solutions) from the produced immunoaffinity columns were in the range from 67 to 100%.