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

Abstract

A simple, efficient and rapid method for the synthesis of cephalosporin-protein conjugates was established. These conjugates were used as immunogens to produce monoclonal antibodies (mAbs) and as solid phase antigens in competitive indirect enzyme immunoassays (EIAs). With this generic approach, a novel set of monoclonal antibodies for cephalosporins was prepared, including ceftiofur and cephalexin as well as, reported here for the first time, cefoperazone, cefquinome and cephapirin. All 5 EIAs were highly sensitive, with standard curve IC(50) values of 0.7 (ceftiofur), 1.1 (cefquinome), 5.2 (cephalexin), 13.8 (cefoperazone) and 40.3 ng mL(-1) (cephapirin). Detection limits (IC(30)) ranged from 0.3 (ceftiofur mAb 1D7) to 17.2 ng mL(-1) (cephapirin mAb 2F10). Specificity studies revealed that cephalosporin-antibody binding was strongly determined by the side chain residues of the cephem nucleus. Therefore all mAbs, to some extent, recognized other beta-lactam antibiotics with similar side chain residues. Within the group of cephalosporins approved for use in veterinary medicine, however, the final EIAs were highly selective for their respective antigen, except for the ceftiofur EIA which showed cross-reactions with cefquinome. The applicability of the five assays for drug residue testing in milk was demonstrated. In each EIA the target drug could be determined in milk with high accuracy and precision at concentrations far below the European Union maximum residue limits.

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

“…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. Positive clones were separated by limiting dilution and subsequently mass produced in miniPERM (Sarstedt, Nümbrecht, Germany) or CELLine (Integra Biosciences AG, Zizers, Switzerland) bioreactors.…”

“…The immunoglobulin subtype of the MAbs was determined by using mouse MAb isotyping reagents purchased from Sigma-Aldrich (St. Louis, MO). The isolation and purification of MAbs were performed by affinity chromatography on protein A or protein G agarose (Sigma-Aldrich), as described before (41).…”

Development and application of monoclonal antibodies against the mycotoxin mycophenolic acid

“…Female mice [BALB/c strain and a hybrid strain of BALB/c×(NZW×NZB)] were immunized with the MPA-KLH conjugate by using established protocols (Bremus et al 2012). Briefly, mice received intraperitoneally 50 μg of the immunogen emulsified in Freunds adjuvant (1+2).…”

Development of a monoclonal antibody-based immunochromatographic strip for cephalexin

“…An ELISA has been developed to detect cephalosporin residues in milk or beef (Bremus, Dietrich, Dettmar, Usleber, & Märtlbauer, 2012;Liang et al, 2012). Based on ELISA, the immunochromatographic assay (ICA) is a more advanced method (Chen, Liu, Kuang, Song, & Xu, 2013;Kuang et al, 2013;Sun et al, 2012;Xie et al, 2009;Xing et al, 2013;Zhao et al, 2011).…”