柴胡皂苷D人工抗原的合成及单克隆抗体的制备

发布时间：2018-01-10 17:21

本文关键词：柴胡皂苷D人工抗原的合成及单克隆抗体的制备　出处：《北京中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的:柴胡是《伤寒杂病论》中应用比较广泛和重要的药物之一,本实验的主要目的是合成柴胡皂苷D(SSD)的人工抗原与人工包被原,制备中药活性成分SSD的单克隆抗体,利用SSD单克隆抗体建立相应的酶联免疫吸附分析方法,检测含柴胡的中药复方中SSD含量,为中药复方质量控制提供新的技术方法,也为后续对经方中柴胡的作用机制和机理的研究奠定基础。研究方法:(1)采用高碘酸钠氧化法合成SSD的人工免疫抗原(SSD-BSA)和人工包被原(SSD-OVA),并且使用薄层色谱法对合成产物进行鉴定;(2)用合成的SSD-BSA多次免疫BALB/c小鼠后,通过ELISA方法测定免疫小鼠抗SSD血清的效价和特异性;(3)取出免疫小鼠的脾细胞与小鼠骨髓瘤细胞SP2/0,采用PEG法进行融合,再进行单克隆细胞株的筛选,采用腹水诱导法生产大量的单克隆抗体;(4)利用制备的SSD单克隆抗体建立ELISA方法,测试该方法的特异性、灵敏度、交叉反应率、回收率、准确性等相关指标;(5)利用建立SSD免疫分析方法,检测五种含有柴胡的复方中SSD含量。研究结果:(1)薄层色谱法鉴定的结果说明,合成的产物SSD-BSA和SSD-OVA偶联成功;小鼠的血清效价在1:10000以上,标准竞争抑制曲线表明,血清中的抗体可与SSD产生特异性结合。取免疫的小鼠脾细胞进行细胞融合,筛选出能分泌抗SSD抗体的单克隆细胞株。基于制备的SSD单克隆抗体,建立相应的ELISA分析法。(2)方法学考察结果显示,在156.25～5000ng/mL范围内,包被原SSD-OVA以1:4000的浓度稀释,腹水以1:100000的浓度稀释,得到SSD单克隆抗体的抑制率与线性关系曲线,回归方程为:y=-0.158In(x)+1.7693,R2=0.9856。IC50为 425.81 ng/mL。回收率在98.15～104.37%(均值为101.19%),板内差异8.16%,板间差异13.78%。通过ELISA法测试SSD与其它中药活性成分反应的交叉反应率。结果显示SSD与其他成分的交叉反应率均小于0.10%。说明制备的SSD单克隆抗体有很好的特异性。研究结论及意义:本实验首次成功的合成了 SSD的人工抗原,为SSD单克隆抗体的筛选和制备以及相应免疫方法的建立奠定基础;首次筛选出SSD的单克隆株;首次制备出SSD的单克隆抗体;首次利用生产出的SSD的单克隆抗体建立酶联免疫吸附分析方法。这些研究可以为中药复方中柴胡的质量监控提供新的检测方法,而且为药物有效成分的分离研究、中药复方有效物质基础的研究、体内代谢、作用机理等的研究奠定基础。
[Abstract]:Objective: Bupleurum chinense is one of the most widely used and important drugs in Typhoid complex Disease. The main purpose of this experiment is to synthesize the artificial antigen and artificial envelope of saikosaponin (DX SSDs). The monoclonal antibody of Chinese medicine active ingredient SSD was prepared and the corresponding enzyme-linked immunosorbent assay (Elisa) method was established by using SSD monoclonal antibody to detect the content of SSD in traditional Chinese medicine compound containing Bupleurum chinensis. To provide a new technical method for the quality control of traditional Chinese medicine compound. It also lays a foundation for further study on the mechanism and mechanism of Bupleurum chinensis in Jingfang. Methods: the artificial immune antigen (SSD-BSA) of SSD was synthesized by sodium periodate oxidation method. And artificial bag coated with SSD-OVA). The synthetic products were identified by TLC. (2) after immunizing BALB/c mice with synthetic SSD-BSA for several times, the titer and specificity of anti-#en3# serum were determined by ELISA method. The spleen cells of immunized mice and mouse myeloma cells SP2 / 0 were taken out and fused by PEG method, then the monoclonal cell lines were screened, and a large number of monoclonal antibodies were produced by ascites induction. (4) the ELISA method was established by using the SSD monoclonal antibody. The specificity, sensitivity, cross reaction rate, recovery rate and accuracy of the method were tested. SSD immunoassay was established to determine the content of SSD in five compounds containing Bupleurum chinense. The synthesized product SSD-BSA and SSD-OVA were successfully coupled. The titer of mouse serum was more than 1: 10000. The standard competitive inhibition curve showed that the antibody in the serum could bind to SSD specifically. The spleen cells of immunized mice were fused to each other. The monoclonal cell lines which can secrete anti SSD antibody were screened. Based on the SSD monoclonal antibody, the corresponding ELISA assay was established. In the range of 156.25 ~ 5000ng / mL, the original SSD-OVA was diluted with 1: 4000 and ascites with 1: 100000. The linear curve of inhibition rate of SSD monoclonal antibody was obtained. The regression equation was 1. 7693. The IC50 of R2N 0.9856. IC50 was 425.81 ng / mL. The recovery rate was 98.15 ~ 104.37 (mean value was 101.19) and the intraplate difference was 8.16%. The cross-reaction rate between SSD and other active components of traditional Chinese medicine was measured by ELISA method. The results showed that the cross-reaction rate between SSD and other components was less than 0.10. The monoclonal antibody to SSD prepared by MMT has good specificity. Conclusion and significance of the study:. In this experiment, the artificial antigen of SSD was successfully synthesized for the first time. It will lay a foundation for the screening and preparation of monoclonal antibodies against SSD and the establishment of corresponding immune methods. The monoclonal strain of SSD was screened for the first time. The monoclonal antibody of SSD was prepared for the first time. Enzyme linked immunosorbent assay (Elisa) was established by using monoclonal antibody of SSD for the first time. These studies can provide a new method for quality control of Bupleurum chinensis. It also lays a foundation for the study of the separation of active components of drugs, the basis of effective substances of traditional Chinese medicine compound, the metabolism in vivo, the mechanism of action, and so on.
【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

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