盐酸克伦特罗抗血清的制备及ELISA检测方法的初步建立

发布时间：2019-01-18 09:19

【摘要】： 为了弥补当前市场上盐酸克伦特罗(CLB)快检方法所存在的缺陷,尤其是以单克隆抗体为基础操作周期长、专业技术要求高等不足之处,本实验利用人工合成抗原BSA-CLB免疫日本大耳白家兔获得了含盐酸克伦特罗抗体的抗血清,并对其进行效价鉴定,在此基础上初步建立了盐酸克伦特罗的酶联免疫吸附检测方法,从而为进一步应用于市场检测奠定了基础。首先,本研究采用正交试验方法,利用重氮化反应原理将CLB和卵清白蛋白OVA、牛血清白蛋白BSA偶联工艺进行优化,分析了各因素对偶联反应的影响机理,并在得出的最佳条件下合成了高偶联率的免疫抗原BSA-CLB和包被抗原OVA-CLB,通过紫外可见全波长扫描分析显示偶联前后载体蛋白的吸收峰发生了很大改变。以经过改进的偶联率计算方法计算得出OVA-CLB和BSA-CLB的偶联率分别为29:1和52.3:1。以免疫抗原BSA-CLB免疫日本大耳白家兔。免疫期间,以OVA-CLB包被酶标板,采用间接非竞争酶联免疫吸附实验检测血清效价,并根据检测结果决定放血时间。免疫四个月后,获得的血清效价最高达到1:640000。最后,本实验利用免疫所得的抗血清,采用间接非竞争ELISA方法比较不同OVA-CLB包被量、不同抗血清及二抗稀释度等对检测结果的影响,确立了最佳检测条件。并在此条件下根据检测标准曲线及灵敏度情况对检测体系的pH值、离子强度、反应时间等进行优化,最终初步建立了以包被OVA-CLB为基础的间接竞争性ELISA检测CLB的方法,检测灵敏度高达0.013μg/mL,标准曲线的线性拟合度高,而且该方法的包被酶标板稳定性、重复性较市售酶标板好,显示出很好的市场应用前景。
[Abstract]:In order to make up for the defects of clenbuterol hydrochloride (CLB) fast detection method in the current market, especially the shortcomings such as long operation cycle based on monoclonal antibody, high technical requirements, etc. The antiserum containing clenbuterol hydrochloride antibody was obtained by immunizing Japanese white rabbits with synthetic antigen BSA-CLB and its titer was identified. An enzyme linked immunosorbent assay (Elisa) for clenbuterol hydrochloride was established, which laid a foundation for further application in market detection. Firstly, the coupling process of CLB and ovalbumin OVA, bovine serum albumin (BSA) BSA was optimized by using the principle of diazotization, and the influence mechanism of various factors on the coupling reaction was analyzed. The high coupling rate of immune antigen BSA-CLB and coated antigen OVA-CLB, were synthesized under the optimum conditions. UV-Vis full-wavelength scanning analysis showed that the absorption peak of carrier protein changed greatly before and after coupling. The coupling rates of OVA-CLB and BSA-CLB are calculated at 29:1 and 52.3: 1, respectively. Japanese large ear white rabbits were immunized with immune antigen BSA-CLB. During immunization, the serum titers were detected by indirect non-competitive enzyme linked immunosorbent assay (Elisa) with OVA-CLB coated with enzyme labeled plates, and the bleeding time was determined according to the results. Four months after immunization, the highest titer was 1: 640000. Finally, by using the immunized antiserum, indirect non-competitive ELISA method was used to compare the effects of different OVA-CLB envelopes, different antiserum and second antibody dilution on the detection results, and the optimal detection conditions were established. Under these conditions, the pH value, ionic strength and reaction time of the detection system were optimized according to the detection standard curve and sensitivity. Finally, an indirect competitive ELISA detection method based on coated OVA-CLB was established. The sensitivity of the method is as high as 0.013 渭 g / mL, and the linear fitting of the standard curve is high. Moreover, this method is more stable and reproducible than that of the marketed enzyme label board, which shows a good prospect of market application.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.1

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