Development and validation of an immunochromatographic assay for rapid multi-residues detection of cephems in milk

Abstract: A one-step immunochromatographic assay (ICA) was developed for the detection of seven kinds of cephems in milk. Polyclonal antibodies (PcAb) with group-specific to cephems were raised in rabbits after immunization with cephalexin–keyhole limpet hemocyanin (KLH) conjugate. The specificity of anti-sera was determined by indirect competitive enzyme-linked immunosorbent assay (icELISA), and the 50% inhibitions (IC50) of cephalexin and cefadroxil were obtained at 1.5ngmL−1; IC50 of cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were 4, 3.7, 3.2, 4.5 and 5ngmL−1, respectively. The PcAb against cephems were conjugated to colloidal gold particles as the detection reagent for ICA strips to test for cephems. This method achieved semi-quantitative detection of cephems in <5min, with high sensitivity to cephalexin and cefadroxil (both 0.5ngmL−1). At the same time, cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were detected at <100ngmL−1 in spiked processed-milk samples. This method was compared with an enzyme-linked immunosorbent assay by testing 40 milk samples, and the positive samples were validated by a high-performance liquid chromatographic method, with an agreement rate of 100% for both comparisons. In conclusion, the method was rapid and accurate for the multi-residue detection of cephems in milk. [Copyright &y& Elsevier]