本文選題：柴胡皂苷c + 單克隆抗體�。� 參考：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：研究目的:(1)合成柴胡皂苷c(SSc)的人工抗原,制備柴胡皂苷c單克隆抗體。(2)在柴胡皂苷c抗體的基礎(chǔ)上建立相應(yīng)的免疫分析方法,并對(duì)該方法進(jìn)行方法學(xué)考察。(3)應(yīng)用建立的免疫分析方法,檢測(cè)中藥復(fù)方中柴胡皂苷c含量,為中藥復(fù)方研究提供新的技術(shù)手段。研究方法:(1)采用高碘酸鈉法制備柴胡皂苷c的包被原(SSc-OVA)和免疫原(SSc-BSA)。利用紫外光譜法和MALDI-TOF-MS法對(duì)SSc-BSA進(jìn)行鑒定。(2)采用背部皮下散點(diǎn)注射的方式對(duì)6周齡雌性BALB/c小鼠進(jìn)行免疫,4次免疫后采用ELSIA法檢測(cè)血清中抗體的效價(jià)及靈敏度。挑選封閉液并篩選包被原工作濃度。采用聚乙二醇(PEG)法融合SP2/0細(xì)胞與免疫鼠脾細(xì)胞,采用有限稀釋法篩選陽(yáng)性單克隆細(xì)胞株。(3)誘導(dǎo)腹水法進(jìn)行抗體的擴(kuò)大化生產(chǎn),并采用辛酸-硫酸銨法進(jìn)行抗體純化。(4)基于SSc的單克隆抗體建立了針對(duì)SSc的競(jìng)爭(zhēng)性ELISA方法,并進(jìn)行了特異性、靈敏度、穩(wěn)定性、回收率等方法學(xué)考察。(5)應(yīng)用SSc的免疫分析法檢測(cè)中藥飲片配制的中藥復(fù)方中柴胡皂苷c的含量及中藥顆粒配制同種復(fù)方中柴胡皂苷c的含量。研究結(jié)果:(1)本研究制備了柴胡皂苷c的人工抗原,采用紫外掃描法和MALDI-TOF-MS對(duì)SSc-BSA進(jìn)行鑒定,SSc-BSA偶聯(lián)成功且偶聯(lián)比為17。(2)篩選SSc-OVA的工作濃度為1:4000,封閉液為5%的脫脂奶粉。SSc-BSA誘導(dǎo)小鼠產(chǎn)生抗SSc血清抗體效價(jià)達(dá)1:16000以上。(3)挑選陽(yáng)性競(jìng)爭(zhēng)均較好的雜交細(xì)胞進(jìn)行單克隆化,多次進(jìn)行傳代、凍存及復(fù)蘇成功獲得性質(zhì)穩(wěn)定的單克隆細(xì)胞株。成功誘導(dǎo)小鼠生產(chǎn)腹水并進(jìn)行純化。ELSIA方法檢測(cè)結(jié)果顯示,純化前后抗體效價(jià)變化不大。(4)本實(shí)驗(yàn)在SSc抗體的基礎(chǔ)上成功建立了 SSc的酶聯(lián)免疫分析方法。篩選包被原及抗體工作濃度,確定SSc-OVA以1:10000包被,純化后抗體稀釋2000倍作為工作液濃度。建立了 SSc的競(jìng)爭(zhēng)抑制曲線,方程為:y=-0.2831n(x)+2.3301,R2=0.9909,線性范圍為156.25-2500ng/mL,靈敏度為625 ng/mL,批內(nèi)變異系數(shù)7.5%,批間變異系數(shù)10%,平均回收率為101.7%,且該ELSIA法且與HPLC相關(guān)性較好。(5)本實(shí)驗(yàn)利用SSc的免疫分析法檢測(cè)了中藥材和中藥顆粒分別配制的大柴胡湯、小柴胡湯等五種中藥復(fù)方中SSc的含量。中藥材配制的柴胡桂枝湯、大柴胡湯、小柴胡湯、補(bǔ)中益氣湯中柴胡皂苷c含量分別為21.91mg/g、16.17mg/g、20.45mg/g、15.06mg/g。中藥顆粒配制的柴胡桂枝湯、大柴胡湯、小柴胡湯、補(bǔ)中益氣湯中柴胡皂苷c含量分別為18.46mg/g、13.41mg/g、17.70mg/g、18.46mg/g。甘草瀉心湯中不含柴胡,故中藥材和中藥顆粒配制的該復(fù)方中均未檢測(cè)到柴胡皂苷c,從側(cè)面驗(yàn)證該ELISA法的特異性。結(jié)論:本實(shí)驗(yàn)采用高碘酸鈉氧化法首次合成了柴胡皂苷c人工抗原,并首次成功制備了柴胡皂苷c的單克隆抗體�；谥苽涞目共窈碥誧單克隆抗體,建立了柴胡皂苷c的酶聯(lián)免疫吸附法。該方法經(jīng)過(guò)方法考察具有穩(wěn)定性好、回收率高等特點(diǎn),可用于柴胡皂苷c的含量測(cè)定。該方法已初步應(yīng)用于中藥復(fù)方成分的檢測(cè)。
[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.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R284

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