Enzyme-linked Immunassay For The Determination Of Allergic Degradation Product Of Benzylpenicillin

β-lactamase was illegally added in milk to degrade penicillin G, in order to escaped from the government inspection of antibiotic, but the allergenic degradation product has the potential hazard to people. To determine the Benzylpenicilloic acid (BPA) in milk, a rapid direct competitive enzyme-linked immunosorbent assay (ELISA) employing a polyclonal antibody generated from benzlypenicillin-KLH was established.Immunogen was artificially produced according to the structures of benzylpenicilloic acid and penicillin G. The anti-Penicillin polyclonal antiserum with a titer of1:240000was obtained which had an inhibition of52.67%to benzylpenicilloic acid (100μg L-1), and was purified by Protein A-Sepharose4B. The established ELISA had an IC50of0.320±0.012μg L-1, and the limit of detection was0.031±0.002μg L-1. The infra-assay and inter-assay were O.85-7.81%and1.33-10.10%separately. The matrix effect of the milk samples were easily eliminated by centrifugation and dilution without any organic solution or clean-up procedure such as SPE or liquid-liquid extraction. Recoveries were between72.75-93.25%.For the validation of the established BPA-ELISA, the milk samples spiked at different concentrations were analyzed by LC-MS. The results showed good correlation between the data of ELISA and LC-MS (R2=0.9977).Different heat treatments of milk have no effects on the established ELISA. Incurred samples got from Tianjin Entry-Exit Inspection and Quarantine Bureau (TJCIQ) were tested by direct competitive ELISA. Almost100%correlation (r2=0.9993) was found compared to the results from TJCIQ.