| In the past few years, there were many incidents about food safety, so the consumersconcerned this problem more than before. The residues of penicillin drugs in milk weredangerous to the healthy of the consumers. The aims of this study are to produce ageneric monoclonal antibody recognizing several penicillins and to develop amulti-residue immunoassay for the detection of these drugs in milk.In this study,6-aminopenicillanic acid was used to synthesize two novel generichaptens of penicillins that were coupled with carrier protein to prepare two immunogensand two coating antigens by using of glutaraldehyde and N, N-carbonyl diimidazoletomethod respectively. The coupling ratios of hapten/carrier were in the range of10-15:1.The two immunogens were used to immunize the Balb/C mice, and then the spleen cellsfrom the mice with the highest serum titer and showing specificity for the hapten werefused with myeloma cells to produce the hybridoma cells. The obtained monoclonalantibody was used to develop an indirect competitive enzyme-linked immunosorbentassay （ELISA）. A checkboard titration method was used to determine the optimalworking concentrations of the coating antigen （1:10000） and the antibody （1:400000）.This method could be used to determine11penicillins （penicillin G, amoxicillin,ampicillin, penicillin V, cloxacillin, oxacillin, cloxacillin, carbenicillin, dicloxacillin,methicillin, nafcillin）. The IC50values were in the range of5.0-36.2ng/mL, thecrossreactivities were in the range of16%-117%and the limits of detection were in therange of0.7-9.3ng/mL. The11drugs were fortified into the blank milk respectively atdifferent levels and the recoveries were in the range of77.6%-99.4%with coefficients ofvariation lower than13.5%.Therefore, the developed ELISA method in this study can be used as a tool for rapiddetection of the residual penicillin drugs in milk with the aim to improve detectionefficiency and ensure the safety of animal original products. |
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