柴胡皂苷c人工抗原的合成及单克隆抗体的制备

发布时间：2018-04-21 15:25

本文选题：柴胡皂苷c + 单克隆抗体　；参考：《北京中医药大学》2017年硕士论文

【摘要】：研究目的:(1)合成柴胡皂苷c(SSc)的人工抗原,制备柴胡皂苷c单克隆抗体。(2)在柴胡皂苷c抗体的基础上建立相应的免疫分析方法,并对该方法进行方法学考察。(3)应用建立的免疫分析方法,检测中药复方中柴胡皂苷c含量,为中药复方研究提供新的技术手段。研究方法:(1)采用高碘酸钠法制备柴胡皂苷c的包被原(SSc-OVA)和免疫原(SSc-BSA)。利用紫外光谱法和MALDI-TOF-MS法对SSc-BSA进行鉴定。(2)采用背部皮下散点注射的方式对6周龄雌性BALB/c小鼠进行免疫,4次免疫后采用ELSIA法检测血清中抗体的效价及灵敏度。挑选封闭液并筛选包被原工作浓度。采用聚乙二醇(PEG)法融合SP2/0细胞与免疫鼠脾细胞,采用有限稀释法筛选阳性单克隆细胞株。(3)诱导腹水法进行抗体的扩大化生产,并采用辛酸-硫酸铵法进行抗体纯化。(4)基于SSc的单克隆抗体建立了针对SSc的竞争性ELISA方法,并进行了特异性、灵敏度、稳定性、回收率等方法学考察。(5)应用SSc的免疫分析法检测中药饮片配制的中药复方中柴胡皂苷c的含量及中药颗粒配制同种复方中柴胡皂苷c的含量。研究结果:(1)本研究制备了柴胡皂苷c的人工抗原,采用紫外扫描法和MALDI-TOF-MS对SSc-BSA进行鉴定,SSc-BSA偶联成功且偶联比为17。(2)筛选SSc-OVA的工作浓度为1:4000,封闭液为5%的脱脂奶粉。SSc-BSA诱导小鼠产生抗SSc血清抗体效价达1:16000以上。(3)挑选阳性竞争均较好的杂交细胞进行单克隆化,多次进行传代、冻存及复苏成功获得性质稳定的单克隆细胞株。成功诱导小鼠生产腹水并进行纯化。ELSIA方法检测结果显示,纯化前后抗体效价变化不大。(4)本实验在SSc抗体的基础上成功建立了 SSc的酶联免疫分析方法。筛选包被原及抗体工作浓度,确定SSc-OVA以1:10000包被,纯化后抗体稀释2000倍作为工作液浓度。建立了 SSc的竞争抑制曲线,方程为:y=-0.2831n(x)+2.3301,R2=0.9909,线性范围为156.25-2500ng/mL,灵敏度为625 ng/mL,批内变异系数7.5%,批间变异系数10%,平均回收率为101.7%,且该ELSIA法且与HPLC相关性较好。(5)本实验利用SSc的免疫分析法检测了中药材和中药颗粒分别配制的大柴胡汤、小柴胡汤等五种中药复方中SSc的含量。中药材配制的柴胡桂枝汤、大柴胡汤、小柴胡汤、补中益气汤中柴胡皂苷c含量分别为21.91mg/g、16.17mg/g、20.45mg/g、15.06mg/g。中药颗粒配制的柴胡桂枝汤、大柴胡汤、小柴胡汤、补中益气汤中柴胡皂苷c含量分别为18.46mg/g、13.41mg/g、17.70mg/g、18.46mg/g。甘草泻心汤中不含柴胡,故中药材和中药颗粒配制的该复方中均未检测到柴胡皂苷c,从侧面验证该ELISA法的特异性。结论:本实验采用高碘酸钠氧化法首次合成了柴胡皂苷c人工抗原,并首次成功制备了柴胡皂苷c的单克隆抗体。基于制备的抗柴胡皂苷c单克隆抗体,建立了柴胡皂苷c的酶联免疫吸附法。该方法经过方法考察具有稳定性好、回收率高等特点,可用于柴胡皂苷c的含量测定。该方法已初步应用于中药复方成分的检测。
[Abstract]:Research purposes: (1) synthesize the artificial antigen of Bupleurum saponins C (SSc) and prepare the monoclonal antibody of Bupleurum saponins C. (2) establish the corresponding immunoassay method based on the antibody of Bupleurum saponins C and investigate the method. (3) the content of Radix Bupleuri saponins C in Chinese medicine compound is detected by using the established immunoassay method, which is a study of Chinese medicine compound. New technical means were provided. (1) the preparation of Bupleurum saponins C by sodium periodate method (SSc-OVA) and immunogen (SSc-BSA). SSc-BSA was identified by ultraviolet spectroscopy and MALDI-TOF-MS. (2) 6 weeks old female BALB/c mice were immunized by subcutaneous injection of the back subcutaneous, and the ELSIA method was used after 4 times of immunization. The titer and sensitivity of antibody in serum were measured. The sealing solution was selected and the original working concentration was screened. PEG was used to fuse SP2/0 cells and immune mouse spleen cells. The positive monoclonal cell lines were screened by the finite dilution method. (3) the antibody was expanded by inducing ascites method and the antibody was purified by octanoic acid ammonium sulfate method. (4) a competitive ELISA method for SSc based on the monoclonal antibody of SSc was established and the specificity, sensitivity, stability and recovery rate were investigated. (5) the content of Bupleurum saponins C in Chinese herbal compound prepared by Chinese herbal decoction and the content of Bupleurum saponins C in the same compound compound were detected by the immunoassay of SSc. The results were as follows: (1) the artificial antigen of Bupleurum saponins C was prepared by using UV scanning and MALDI-TOF-MS to identify SSc-BSA, SSc-BSA coupling was successful and the coupling ratio was 17. (2), the working concentration of SSc-OVA was 1:4000, and the anti SSc serum antibody titer of.SSc-BSA induced mice was 5%. (3) The hybrids with better positive competition were McAb, and the monoclonal cell line was successfully obtained for several times. The results of successfully inducing mice to produce ascites and purifying the.ELSIA method showed that the antibody titer changes before and after the purification were not great. (4) the experiment was successfully established on the basis of SSc antibody. The enzyme linked immunosorbent assay (ELISA) of SSc was used to screen the original and antibody working concentration, to determine that SSc-OVA was coated with 1:10000, and the purified antibody was diluted 2000 times as the working fluid concentration. The competition inhibition curve of SSc was established. The equation was y=-0.2831n (x) +2.3301, R2=0.9909, the linear range of 156.25-2500ng/mL, the sensitivity of 625 ng/mL, and the intra variation system. The coefficient of variation was 7.5%, the coefficient of variation was 10%, the average recovery rate was 101.7%, and the ELSIA method had a good correlation with HPLC. (5) this experiment used the immunoassay of SSc to detect the content of SSc in five kinds of Chinese medicine, such as Dai Hu soup, Xiao Chai Hu soup, etc., respectively. The content of Bupleurum saponins C in Buzhong Yiqi soup is 21.91mg/g, 16.17mg/g, 20.45mg/g and 15.06mg/g., respectively. The content of Chaihu Guizhi soup, Dai Hu soup, small Bupleurum soup, and buchaihu saponins C content are 18.46mg/g, 13.41mg/g, 17.70mg/g and 18.46mg/g. Glycyrrhiza Xiexin soup, so the Chinese medicine and traditional Chinese medicine are not contained. The radix Bupleurum saponins C was not detected in the compound, and the specificity of the ELISA method was verified from the side. Conclusion: the artificial antigen of Bupleurum saponins C was synthesized by the oxidation of sodium periodate for the first time, and the monoclonal antibody of the saponins C was successfully prepared for the first time. Based on the preparation of the monoclonal antibody against Radix Bupleuri saponins C, the soaps were established. The enzyme linked immunosorbent assay (ELISA) of glucoside C has the advantages of good stability and high recovery. It can be used for the determination of the content of C in Bupleurum saponins. This method has been preliminarily applied to the detection of compound ingredients in Chinese medicine.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284

【参考文献】