Preparation Of Monoclonal Antibodies Against Ampicillin And Development Of ELISA Test Kit

Ampicillin (Amp) was combined with mcKLH by the method of EDC intermediate, then the combination injected Balb/c mice as immunogen. The combination, Amp-cBSA, which obtained by combined Amp and cBSA according to Amp-KLH method, was used to detect the hybridomas for secreting antibody against Amp. The coupling ratio of Amp to BSA was 10:1 and to KLH was 30:1. Balb/c mice were immuned by the immunogen and the titer of anti-serum was 2×105. The hybridomas were obtained by fusing mouse myeloma cells SP2/0 with splenocytes from the mice immunized with Amp-KLH. Eight strains of hybridoma cells were obtained with secreting anti-Amp antibody, named 2A6,2G6,2A10,2B8,3D12,3E12,4E1 and 4D12. The ascites of McAbs were prepared by injecting the hybridoma cells into mice abdomen. The indirect ELISA results showed that the McAbs revealed high affinity with Amp at the titer of 2×106. The purified McAbs can be used in the detection of the Amp residue. The characteristics of monoclonal antibody was performed by ELISA test, chromosomal counting and SDS-PAGE electrophoresis. The results indicated that the subclass of the McAb was IgG2a, the chromosomal numbers were 97-104, the affinity constant was 3×10-10mol/L, the molecular weight was 154KD. The cross-reactivities of McAb to ampicillin, ampicillin-Na, penicillin-Na were 100%, 0.36%, 0.0065%, respectively, and to cefradine, ceftiofur, kanamycin, streptomycin, sulfadiazine, gentamicin were all below 0.001%. The indirect competitive Enzyme Linked Immunosorbent Assay(ELISA) for Amp was developed. The best concentration of coating antigen was 2μg/ml, the optional working dilution of the McAb was 1:50000 and enzyme conjugate antibody was 1:5000,The linear detecting range of calibration curves to detect Amp was 0.5ng/ml～100ng/ml.The formular was Y=0.0569X+0.1308, R2=0.9948. The limit of detection was 0.3ng/ml, the detection limit was 0.4 ng/ml in milk.