Studies On Immunological Rapid Determination Of Difloxacin Residues

In the study, Based on the analysis of molecular structure and immunogenicity of Difloxacin, the technology of monoclonal antibodies production was applied to assemble rapid ELISA kit and strip for DIF residual detection. The main contents and results of this study were as follows:1. Synthesis and identification of the artificial antigen for difloxacinDifloxacin was coupled with carrier protein bovine serum albumin(BSA) and ovalbumin(OVA) to prepare complete antigen DIF-BSA and DIF-OVA by modified method of carbodiimide. DIF-BSA and DIF-OVA were synthesized successfully and their conjugation ratio of DIF to BSA and OVA respectively were about 4.7:1 and 4.8:1 by identification with UV scanning spectrum and SDS-PAGE, the high-titer, sensitive and specific DIF antiserum of Balb/C mice immunized with DIF-BSA had been produced by identification with indirect ELISA.2. Preparation of monoclonal antibodies and immunological characterization of DifloxacinOne Balb/C mice were chosen from three Balb/C mice immunized with DIF-BSA by the titer of indirect ELISA and blocking ELISA after fourth immunization. The hybridoma lines that secrete DIF mAb were established with using hybridoma technology. The immunological traits such as titer, affinity and specificity of the mAb were characterized. The results showed that three hybridoma cell lines of 1H10, 2F12 and 4D8 were screened for specificity to DIF, the indirect ELISA titer of the mAb were 1:5.12×102, 1:3.2×101 and 1:2.56×102 in supernatant, 1:1.6×105, 1:4×104, 1:8×104 in ascites, the isotypes of the mAb were IgG1, IgG1, IgG2a, and the affinity constant(Ka) were 2.94×1010 L/mol, 1.72×1010 L/mol and 1.35×1010 L/mol, respectively, the mAb of 1H10 showed good sensitivity with IC50 of 15.6 ng/ml to DIF. mAb has no cress-reactivity to other FQS and sulfonamides. DIF mAb of high-titer, sensitivity and specificity had been generated, it is possible to establish immunoassay of DIF residues in animal food.3. Development of rapid determination of Difloxacin residue method Based on the enzyme-linked immunosorbent assay principle, a competitive ELISA kit for determination of DIF (DIF-Kit) was developed with DIF mAb. The calibration curve of DIF-Kit with standard DIF inhibitor was typical sigmoid curve fitted to the four parameters logistic equation with the linear determination of 1.0 to 128.0 ng/ml, the detection limit for DIF was 0.1 ng/ml and IC50 was 2.2 ng/ml. The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15% in range of 0.1-128.0 ng/ml. The recoveries from milk and chicken were 89.3% and 83.9%, respectively, when 0.4, 2.0, 10.0, 50.0 ng/ml DIF were spiked. The validity of DIF-Kit in 4℃was above six months.Basis on principle of gold immunochromatography assay (GICA), a rapid test strip of DIF (DIF-Strip) was developed with DIF mAb. In practice the lower limit of detection using a strip reader was 0.5 ng/ml and by unaided visual assessment 2 ng/ml. Samples of milk were spiked at 2 to 8 ngDIF/ml, gave recoveries of between 94.5 and 107% and the coefficient of variation (CV (%)) 1.6-9 %. The test of DIF residues could be accomplished within 60 minutes by DIF-Kit and within 8 to 10 minutes by DIF-Strip. The data suggest that the kit and test strip meets the requirements of high sensitivity, specificity, simplicity and speed of performance, they are suitable for the determination of DIF residues rapidly and reliably in a quantitative, semi-quantitative or qualitative mode.