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 beta-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 (IgG2a) had marked cross-reactivities with most of the 17 tested beta-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 micrograms 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%.

Enzyme-Linked Immunosorbent Assay of Ampicillin in Milk

“…The literature is replete with methods for obtaining poly-or monoclonal antibodies against penicillins with closed [10,19,[21][22][23] or open [9,11,[18][19][20]23] β -lactam rings (group I and II immunogens, respectively). Preparation of group I immunogens involves their conjugation with proteins.…”

“…Preparation of group I immunogens involves their conjugation with proteins. Several methods have been published in which ampicillin is conjugated via its amino group [19,[21][22][23]; in addition, both ampicillin [19] and cloxacillin [10] may be conjugated via the carboxyl of the thiazolidine ring using the method of mixed anhydrides. Group II immunogens are obtained as described below.…”

A broadly applicable approach to prepare monoclonal anti-cephalosporin antibodies for immunochemical residue determination in milk

“…Finally, 3 days before fusion, mice exhibiting both high serum antibody titres and high antibody affinity for free antigen, received a final booster injection of 75 μg of antigen in 300 μL PBS alone. Cell fusion using X63-Ag8.653 myeloma cells was performed as previously described [34]. Culture supernatants were tested for cephalosporin-specific antibodies 12 days after fusion in non-competitive indirect EIAs with the respective cephalosporin-protein conjugate as the solid phase.…”

Simultaneous Rapid Detection and Serotyping of Cronobacter sakazakii Serotypes O1, O2, and O3 by Using Specific Monoclonal Antibodies

“…The preparation of protein and LPS extracts by treating bacterial strains with polymyxin B-sulfate and the immunization of female mice [BALB/c strain and a hybrid strain of BALB/c ϫ (NZW ϫ NZB)] was conducted as described before (39). The fusion of splenocytes and X63-Ag8.653 myeloma cells was performed as published by Dietrich et al (40). The culture supernatant of the hybridoma cells was screened for serotype-specific antibodies using a standard EIA protocol (39,41), with concentrated bacterial preparations serving as the solid phase.…”