本文選題：柴胡皂苷c + 單克隆抗體��； 參考：《北京中醫(yī)藥大學》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抗體的基礎上建立相應的免疫分析方法,并對該方法進行方法學考察。(3)應用建立的免疫分析方法,檢測中藥復方中柴胡皂苷c含量,為中藥復方研究提供新的技術手段。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SSc-BSA進行鑒定。(2)采用背部皮下散點注射的方式對6周齡雌性BALB/c小鼠進行免疫,4次免疫后采用ELSIA法檢測血清中抗體的效價及靈敏度。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細胞與免疫鼠脾細胞,采用有限稀釋法篩選陽性單克隆細胞株。(3)誘導腹水法進行抗體的擴大化生產(chǎn),并采用辛酸-硫酸銨法進行抗體純化。(4)基于SSc的單克隆抗體建立了針對SSc的競爭性ELISA方法,并進行了特異性、靈敏度、穩(wěn)定性、回收率等方法學考察。(5)應用SSc的免疫分析法檢測中藥飲片配制的中藥復方中柴胡皂苷c的含量及中藥顆粒配制同種復方中柴胡皂苷c的含量。研究結果:(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SSc-BSA進行鑒定,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誘導小鼠產(chǎn)生抗SSc血清抗體效價達1:16000以上。(3)挑選陽性競爭均較好的雜交細胞進行單克隆化,多次進行傳代、凍存及復蘇成功獲得性質穩(wěn)定的單克隆細胞株。成功誘導小鼠生產(chǎn)腹水并進行純化。ELSIA方法檢測結果顯示,純化前后抗體效價變化不大。(4)本實驗在SSc抗體的基礎上成功建立了 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的競爭抑制曲線,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相關性較好。(5)本實驗利用SSc的免疫分析法檢測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方中均未檢測到柴胡皂苷c,從側面驗證該ELISA法的特異性。結論:本實驗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)過方法考察具有穩(wěn)定性好、回收率高等特點,可用于柴胡皂苷c的含量測定。該方法已初步應用于中藥復方成分的檢測。
[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.

【學位授予單位】：北京中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R284

【參考文獻】

相關期刊論文 前10條

1 查政;;柴胡桂枝湯在神志病治療中的運用及機理探討[J];國醫(yī)論壇;2017年02期

2 王蓓;梁志菊;;柴胡桂枝湯加減方治療病毒性心肌炎臨床療效觀察[J];四川中醫(yī);2017年01期

3 楊曉靈;廖小英;黃媛;潘清清;黃麗英;;SFOD-LPME-HPLC測定金線蓮中對羥基苯乙酮和香草乙酮[J];中國醫(yī)院藥學雜志;2016年24期

4 孫冬梅;陳秋谷;李養(yǎng)學;李素梅;;清熱疏肝顆粒的質量標準研究[J];廣東藥學院學報;2016年05期

5 閆婕;衛(wèi)瑩芳;龍飛;林波;康敏;;馬爾康柴胡不同藥用部位解熱、保肝作用與急性毒性研究[J];世界科學技術-中醫(yī)藥現(xiàn)代化;2016年08期

6 楊曉泉;夏從龍;;一測多評法測定獐牙菜屬植物中4種環(huán)烯醚萜類成分[J];亞太傳統(tǒng)醫(yī)藥;2016年15期

7 馮偉紅;李春;張鍇鑌;楊立新;易紅;陳兩綿;張永欣;榮立新;王智民;;“一測多評”模式在淫羊藿藥材、飲片及含淫羊藿中成藥黃酮類成分檢測中的一體化研究[J];中國中藥雜志;2016年15期

8 焦秋偉;孟利;孫歡歡;;單克隆抗體制備的研究進展與展望[J];內江科技;2016年06期

9 迪更妮;張維庫;喬灝yN;屈會化;趙琰;王慶國;續(xù)潔琨;;酸棗仁皂苷A人工抗原的制備及鑒定[J];中國中藥雜志;2016年10期

10 胡驍飛;鄧歌;柴書軍;姚靜靜;孫亞寧;王方雨;鄧瑞廣;張改平;;玉米赤霉醇完全抗原的制備及質量鑒定[J];西北農(nóng)業(yè)學報;2016年05期

相關會議論文 前1條

1 李曉宇;李曉驕陽;孫蓉;;柴胡皂苷a對人肝細胞L-02的體外肝毒性機制研究[A];2013年中國藥學大會暨第十三屆中國藥師周論文集[C];2013年

相關博士學位論文 前7條

1 姚敏;柴胡皂苷d對人前列腺癌DU145細胞的增殖抑制和凋亡誘導作用及機制研究[D];吉林大學;2016年

2 杜淑媛;水產(chǎn)品中危害物膠體金免疫層析毛細管檢測技術的研究[D];中國海洋大學;2015年

3 于歡;鱉血柴胡與雄黃顆粒飲片的炮制現(xiàn)代研究[D];北京中醫(yī)藥大學;2015年

4 李鑫;基于免疫分析的農(nóng)產(chǎn)品真菌毒素混合污染同步檢測技術研究[D];中國農(nóng)業(yè)科學院;2014年

5 張中曉;柴胡毒素毒性代謝組學及毒性機制研究[D];第二軍醫(yī)大學;2014年

6 王硯;竹葉柴胡和北柴胡品質比較研究[D];成都中醫(yī)藥大學;2014年

7 張靜;川楝素免疫分析方法及其應用研究[D];西北農(nóng)林科技大學;2008年

相關碩士學位論文 前10條

1 迪更妮;中藥山茱萸和酸棗仁中馬錢苷、酸棗仁皂苷A人工抗原的合成及多克隆抗體的制備[D];北京中醫(yī)藥大學;2016年

2 孫慧敏;柴胡醋制前后的化學及藥理比較研究[D];山西大學;2015年

3 李壯;嘌呤生物堿類化合物茶堿-7-乙酸、theacrine和咖啡因的ELISA方法的建立及應用[D];北京中醫(yī)藥大學;2015年

4 成金俊;淫羊藿苷單克隆抗體的制備及其ELISA法的建立和FLISA法的初探[D];北京中醫(yī)藥大學;2015年

5 陳麗娜;柴胡皂苷吸收、聚集特性及隱性柴胡皂苷的研究[D];河南科技大學;2015年

6 朱玉嬋;免疫膠體金技術在鴨坦布蘇病毒和快速定量檢測Asial型口蹄疫病毒的應用[D];河北農(nóng)業(yè)大學;2014年

7 劉進鍇;柴胡皂苷d對大鼠肝星狀細胞HSC-T6的增殖活化及雌激素受體表達的影響[D];第二軍醫(yī)大學;2014年

8 陳利;中藥柴胡主要活性成分柴胡皂苷D作用于肝細胞LO2的肝損傷機理研究[D];南京中醫(yī)藥大學;2014年

9 張倩;柴胡皂苷結構轉化及柴胡皂苷b_2毒性初步研究[D];河南科技大學;2014年

10 王梅;北柴胡不定根培養(yǎng)高效合成柴胡皂苷研究[D];山西大學;2013年

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