| Enzyme-linked immunosorbent assay (ELISA) was developed for analysing oxytetrocycline(OTC) from the bee products using the anti-OTC antibody. Main research contents were followed:1 Preparation for OTC-BSAOTC is hapten, which only has reactiongenicity but no immunogenicity, due to its low molecule weight (460.2). It may be changed into complete antigen, which can stimulate animal to produce antibody, when OTC is conjugated with carrier-protein such as bovine serum albumin(BSA), ovalbumin(OV) etc. OTC can be directly conjugated with BSA by amidogen in OTC as 'bridge'.EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and GA (glutaraldehyde) were used as adjuncts to conjugate Oxytetracycline to BSA and OV respectively. Both EDC and GA could conjugate OTC to BSA by UV spectrum analysis. However, the molar ratio of OTC to BSA in artificial antigen was 4.00:1 (using EDC as adjunt) and 2.85:1 (GA as adjunt) respectively. This means EDC is better adjunct than GA in preparation for OTC-BSA.Then the effects of the different mix method of protein, OTC and EDC on the molar ratioes of OTC to BSA in the artificial antigen were studied, that results showed that adding OTC and EDC to BSA drop by drop was better than adding them directly. The OTC-BSA was good for stimulating mouse to produce antibody against OTC.2 Production of anti-OTC antibodyBALB/c mice were injected with the OTC-BSA. The antibody began to obviously produce in mouse when the conjugate was injected after 45 days, and 90 days reached to peak, then began to go down. The highest titer of antibody at peak was 1:10,000, 1:5,000 and 1:20,000 from mouse NO. 1, mouse NO.2 and mouse NO.3 respectively.The sensitivities and specificities of the antibody were analyzed using indirect competitive ELISA. The results revealed that the antibodies have good sensitivities and specificities and low ratio of cross reactivities with other structure similar antibiotics suchas penicillin, streptomycin and terramycin.3 ELISA detection of OTC residue from bee productsIn this research, the the extracting conditions and methods of OTC from honey, pollen and honey bee were studied, and then the best extracting method and conditions were obtained. On the basis of these, the indirect competitive ELISA (ICELISA ) was used to analyze the OTC content in bee products. The results showed that the detecting range was 5~200ng/ml, and the recovery of this method was 97.5%~107%.4 Preparation for ELISA kitsELISA kits were stored at 4°C for two months and -20 °C for three months after appropriate pre-treatment, the OD49onm values of kits were detected by indirect ELISA, the results revealed that the OD49onm values of kits were 1/10 and 1/3 times of initial value, respectively. |
