【作者】 邢淑婕；

【导师】 陈福生；

【作者基本信息】 华中农业大学 ， 微生物学， 2005， 硕士

【摘要】 本研究通过制备土霉素(oxytetracycline, OTC)的特异性抗体,建立了蜂产品中土霉素残留的酶联免疫吸附分析法(enzyme-linked immunosorbent assay, ELISA)。具体包括以下四个方面的内容: 1 土霉素人工抗原的构建 OTC分子量为460.2,是只有反应原性而无免疫原性的小分子物质,必须与蛋白质等载体结合转化为完全抗原后才能刺激动物产生抗体。由于OTC分子结构中含有氨基,所以可以以该基团为“桥梁”将OTC和载体蛋白连接起来。 本研究首先分别以碳化二亚胺(1-ethy 1-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDC)和戊二醛(commercial glutaraldehyde, GA)作为偶联剂,将OTC与牛血清白蛋白(bovine serum albumin, BSA)进行偶联,合成了复合物OTC-BSA,并分析了不同连接剂对蛋白偶联效果的影响。结果表明,以EDC或GA为偶联剂,均可得到OTC与BSA的偶联物,但是偶联物中OTC与BSA的摩尔比有很大的差异,以EDC为偶联剂时OTC:BSA摩尔比为4.00:1,而以GA作为偶联剂时仅为2.58:1,这说明EDC的偶联效果比GA的好。 进一步就OTC与BSA偶联时,BSA、OTC及偶联剂的混合方法对偶联物摩尔比的影响进行了研究,结果显示,向BSA溶液中边搅拌边缓慢地滴加OTC和EDC溶液时OTC-BSA偶联物中OTC与BSA的摩尔比较高,达7.97:1。该连接物注射BALB/c小鼠后能获得较好的抗体。 本研究还在上述研究的基础上制备了OTC与卵清白蛋白(ovalbumin, OV)的偶联物OTC-OV。 2 抗OTC抗体的制备 以自制的OTC-BSA(摩尔比为7.97:1)连接物为免疫抗原注射BALB/c小白鼠,从抗体的产生进程来看,小鼠在第一次免疫后45天开始有明显的抗体产生,90天达到高峰,然后开始下降,间接非竞争ELISA测得其最高效价为1:20,000;进一步用间接竞争ELISA分析了此抗体的灵敏度与特异性,结果表明,所得抗体的灵敏度和特异性都较好,和其它结构相似或相近物质的交叉反应率较低。 3 几种蜂产品中OTC的ELISA检测 对蜂蜜、蜂花粉、蜂王浆中OTC的提取方法进行了研究,得出了它们的最佳提更多还原

【Abstract】 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.更多还原