Preparation Of Antibodies And Establishment Of ELISA Methods For Ofloxacin And Difloxacin

In this study, ofloxacin(OFL) and difloxacin(DIF) were linked respectively to two kinds of carrier proteins ,by which the four kinds of artificial antigens were synthesized successfully. Using the artificial immunogens, rabbits were immunized to obtain the antiserum against ofloxacin, the antiserum against difloxacin and the antiserum against ofloxacin and difloxacin.The Ci-ELISA methods for the detection of ofloxacin were established with the antiserum against ofloxacin and the antiserum against ofloxacin and difloxacin.And the Ci-ELISA methods for the detection of difloxacin were established with the antiserum against difloxacin and the antiserum against ofloxacin and difloxacin .1.Synthesis and Identification of the Artifical Antigens for Ofloxacin and Difloxacin.Ofloxacin and difloxacin were coupled respectively to bovine serum albumin (BSA) by Carbodiimide method, Ofloxacin and difloxacin were also coupled respectively to ovalbumin(OVA) by Carbodiimide method.by which the Conjugates OFL-BSA, OFL-OVA,DIF-BSA and DIF-OVA were prepaired.The Conjugates were identified by colour reaction of FeCl3 and UV Spectrum.The results demonstrated that the four kinds of artificial antigens were synthesized successfully.The Conjugating ratios of the artifical Antigens were calculated by UV Spectrum method.The results were as follows:The Conjugating ratios of OFL-BSA and OFL-OVA were 11.16:1 and 5.90:1,respectively. The Conjugating ratios of DIF-BSA and DIF-OVA were 5.47:1 and 6.40:1,respectively.2.Preparation of Antisera and Determination of Working Concentrations for ELISA.DIF-BSA,OFL-BSA and the mixture of them were used as immunogens to immunise rabbits,by which the antiserum against ofloxacin, the antiserum against difloxacin and the antiserum against ofloxacin and difloxacin were obtained.The titers of the antiserum against ofloxacin and the antiserum against difloxacin were both 1:2~9×10~4.Using OFL-OVA as coating antigen, the titer of the antiserum against ofloxacin and difloxacin was 1:2~9×10~4. Using DIF-OVA as coating antigen, the titer of the antiserum against ofloxacin and difloxacin was 1:28×104. Using the antiserum against ofloxacin and the antiserum against ofloxacin and difloxacin respectively to establish Ci-ELISA methods for ofloxacin ,the working concentrations of the coating antigen (OFL-OVA) were 2μg/ml and 0.25μg/ml respectively, the working concentrations of the antisera were both 1:4×10~5. Using the antiserum against difloxacin and the antiserum against ofloxacin and difloxacin respectively to establish Ci-ELISA methods for difloxacin ,the working concentrations of the coating antigen(DIF-OVA) were 0.25μg/ml and 1μg/ml respectively, the working concentrations of the antiserum against difloxacin and the antiserum against ofloxacin and difloxacin were 1:2×10~5 and 1:4×10~5 respectively.3. Establishment of Ci-ELISA methods for Ofloxacin and DifloxacinOne standard curve of ofloxacin was estabished with the antiserum against ofloxacin.the regression equation, the 50% inhibition concentration(IC50), the limits of detect (LOD) and the linear range about the standard curve were y=-0.2044x+0.9610 (r=0.9927), 180.32ng/ml,0.85ng/ml and 0.85~10000ng/ml, respectively. Another standard curve of ofloxacin was estabished with the antiserum against ofloxacin and difloxacin, the regression equation, the IC50, the LOD and the linear range about the standard curve were y=-0.1984x+0.9687(r=0.9885), 231.57ng/ml,1.62ng/ml and 1.62~10000ng/ml, respectively. One standard curve of difloxacin was estabished with the antiserum against difloxacin, the regression equation, the IC50, the LOD and the linear range about the standard curve were y=-0.223x+0.9631(r=0.9929) , 119.21ng/ml,1.14ng/ml,1.14~10000ng/ml,respectively. Another standard curve of difloxacin was estabished with the antiserum against ofloxacin and difloxacin, the regression equation, the IC50, the LOD and the linear range about the standard curve were y=-0.1738x+0.915(r=0.9985), 245.32ng/ml, LOD=1.86ng/ml, 1.86~10000ng/ml, respectively. The above results suggested that all the standard curves had good linear relations in their linear ranges,the sensitivities of the Ci-ELISA methods which had been estabished in this study were satisfying for detecting ofloxacin or difloxacin.The coefficients of variation for all the Ci-ELISA Methods which had been estabished in this study were all low 7% in intra-assay and 15% in inter-assay. The recoveries of ofloxacin and difloxacin in milk were detected ,The results showed that the recoveries of all the Ci-ELISA methods ranged from 71.70% to 92.85%. So the coefficients of variation and the recoveries accord with the requests of ELISA.Some antibiotics were selected to do the experiments of cross-reaction.The results revealed that the antisera had cross-reactivity of differant degrees with the fluoroquinolones which were selected in this study,but they had no cross-reactivity with sulfadiazine, chloramphenicol, terramycin or penicillin.