Rapid Immunological Determination Of Enrofloxacin Residues

Enrofloxacin (ENR) is a synthetic antibacterial agent that belongs to the fluoroquinolone group, and it has been increasingly used in veterinary medicine to treat microbial infections. But enrofloxacin residues in edible animal tissues raise concerns for public health. Because of concerns about drug residues entering the food chain and contributing to bacterial resistance, some countries have established the MRL for drugs. Although classical analytical methods have been described for the detection of enrofloxacin, including HPLC, LC-MS, these methods require extensive sample preparation as well as highly trained individuals to operate sophisticated instruments, are not suitable for use in the field. To test samples under working conditions, rapid and inexpensive screening methods are required., the immunological analytical technique, which has many merits of sensitiveness, specificity, rapidity and convenience, have been generally used in determination of drug residues. This study aimed at immunological rapid determination of ENR residues, including the immunogenicity of ENR, synthesis of artificial antigen, preparation and special properties of ENR pAb, ENR mAb, reagent kits, immunological gold-labeled strip, and confirmed by LC-MS.1. Basis on analysis of molecular structure and immunogenicity of ENR, immunogen and covering antigen were prepared by active ester method and isobutyl chloroformade method coupling ENR to BSA or OVA. By the ultraviolet absorb spectral curve and SDS-PAGE electrophoresis, hapten ENR was coupled successfully to carrier protein, and conjugation- ratio was different in different methods or different conditions. To immunize Balb/C mice using ENR-BSA(1∶29, 1∶16, 1∶9), antibody titer and IC50 were also different. From the above, the best mouse was choosed to cell fusion for the monoclonal antibody preparation.2. Three New Zealand white rabbits were immunized with BSA-ENR and ENR pAb had been produced. The ENR pAb had the high titers of 1∶2.56×105 by indirect ELISA, a good sensitivity of IC50 (11μg/L) and cross-reactivity(CR) to other compounds was <0.1% by blocking ELISA. The ENR pAb had a good specificity by ELISA and West-Gold.3. Balb/C mice were chosen from nine Balb/C mice immunized with BSA-ENR for cell fusion by the titer of indirect ELISA and blocking ELISA. Two hybridoma lines of 4G1-B3, 4G1-G1 that secrete ENR mAb were screened by indirect ELISA and blocking ELISA. The indirect ELISA titers of them were 1 : 1280, 1 : 640 in supernatant, 1 : 1.024×106, 1 : 5.12×105 in ascites respectively, the isotype of them was IgG1 and the affinity constant(Ka) was 9×1010, 4.5×1010 (L/mol) respectively. The best one of them was 4G1-B3, ENR mAb of 4G1-B3 showed good sensitivity with an IC50 of 1.59μg/L to ENR and CR% to other compounds was <0.01% by blocking ELISA, specificity integrated with BSA-ENR that determined by ELISA and West-Gold assay. ENR pAb and ENR mAb obtained were both used to establish immunoassay of ENR residues, but ENR mAb was better than ENR pAb.4. A blocking ELISA kit and a competitive ELISA kit for determination of ENR (ENR -Kit) were developed with ENR mAb of 4G1-B3, the competitive ELISA kit was better than the blocking ELISA kit by comparison. The calibration curve of ENR -Kit with standard ENR inhibitor was typical sigmoid curve fitted to the four parameters logistic equation with the linear determination of 0.053 to 101.6μg/L, the sensitivity of 0.053μg/L and the determination limit of 0.25μg/L, IC50 was 1.1μg/L. The recoveries of ENR spiked in milk were 97.25%, in chicken were 84.28%, in fish were 85.95%. The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15%. The CR% to other compounds was <0.01%. The dilution solution of ENR had no effect on results of ENR-Kit. The validity of ENR-Kit in 4℃was above six months.5. Basis on principle of gold immunochromatography assay (GICA), a rapid test strip of ENR (ENR-Strip) was developed with ENR mAb. The calibration curve of ENR-Strip was typical sigmoid curve fitted to the four parameters logistic and its determination limit was 0.2μg/L by BioDot-TSR3000, 1.0μg/L by eyes. The CR% to other compounds was <0.1%. The validity of ENR-Strip in 4℃was above six months.6. The sensitivity, specificity, simplicity and reliability of ENR-Kit and ENR Strip were confirmed by the procedure utilizing LC-MS for the identification and quantification of ENR in chicken. Their coincidence rate was 100% with LC-MS. The kit and strip were proved to be used for the rapid determination of ENR residues.