| Chloramphenicol(CAP)is the crucial residues in animal foodstuff,so that it is urgent to establish accurate screening methods.Enzyme-linked immunosorbent assay(ELISA)is a convenient,quick,highly efficient way and is suitable for the on-site detection of the veterinary Drugs residues in animal foodstuff.In this research,we synthesized and screened highly efficient antigen,developed a monoclonal antibody(mAb)by further screening after cell fusion,and then,we developed ci-ELISA followed by the mAb.Firstly,we synthesized the immunogen CAP-HS-BSA and the coating antigen CAP-HS-OVA with mixed anhydride method,characterized the antigen with UV-vis and SDS-GAGE,Compared with BSA alone,the absorption peak of CAP-HS-BSA shifted in UV spectrum,and CAP-HS-BSA migrated slower in SDS-PAGE,indicating that CAP-HS-BSA has been conjugated successfully.The conjugation ratio of CAPHS with BSA was calculated as 12.1:1.Immunized and screened the best immunogen,the proper detect antigen and the best mice by ELISA.Indirect ELISA showed that the titers of the anti-CAP polyclonal antibody(CAP mAb)were greater than 1:6.4×103.The pAb from one mouse gave the best IC50 of 16.4 ?g/L by indirect competitive ELISA(ci-ELISA),and has not cross reaction(CR)to the other compounds.These data indicated that pAb against CAP with high-titer,specificity and sensitivity was obtained.MAb to CAP was produced by fusing spleen cells from the immunized mouse with SPNS0 cells.Three hybridoma cell lines of 1B8 CAP mAb?2C4 CAP mAb and4A1 CAP mAb were screened out using indirect and ci-ELISA.Indirect ELISA showed the titers of these four positive hybridoma cells were 1?2.4×102?1?6.4×102?1?1.2×102 in the supernatant,and were 1?2.0×105?1?4.8×105?1?1.2×105 in ascites,respectively.2C4 shows the best IC50 of 0.53?g/L to CAP and has noCR to other compounds.A ci-ELISA was established using the mAb 2C4,and based on the competitive binding of free CAP in the sample and the coated CAP-BSA with the 2C4.The optimal conditions for ci-ELISA were determined by chessboard ELISA as the followings: coating concentration of CAP-HS-OVA = 0.4 ?g/mL;the working concentration of 2C4 = 1:6.4×104;the diluted concentration of Go?MoIgG-HRP =1:1×103;the plate was coated with 50 ?L of CAP-OVA at 37 °C for 120 min,blocked for 60 min with 5% porcine serum at room temperature(RT).TMB was used for color development at RT for 9 min.The calibration curve of ci-ELISA CAP showed typical sigmoid and fitted to the four parameters logistic equation.The IC50 in ci-ELISA was0.53 ?g/L and the limit of detection was 0.6 ?g/L.The average recoveries of CAP in negative carp meat and pork were 97.3% and 97.5%;the average coefficient variation were 4.9% and 5.5%,and both were lower than 10%.The CAP-Kit generally has not CR to other compounds.HPLC comparison: Ci-ELISA measurement results and differences in sample value is 3.30%~4.40%,Ci-ELISA and HPLC of the difference is1.13%~4.85%;the determination of practical samples:,the differences of Ci-ELISA and HPLC was 4.43% in shrimp samples. |
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