本文選題：β2微球蛋白　切入點：單克隆抗體　出處：《鄭州大學》2014年碩士論文

【摘要】：β2微球蛋白（β2-microglobulin，β2-MG）是由機體產生的一種內源性低分子量血清蛋白質，分子量11.8KD，是細胞膜上完整主要組織相容性復合物（majorhistocompatibility complex，MHC）Ⅰ類分子的輕鏈（β鏈），在免疫應答中起重要作用。隨著代謝和MHC的降解，β2微球蛋白分離后以游離形式存在于血清、尿液、腦脊液、唾液等體液中，含量較低且恒定。近年來，β2微球蛋白在基礎醫(yī)學及臨床診斷上的應用已成為研究熱點，檢測血清或尿液中β2微球蛋白含量的變化成為評價相關臟器功能和某些疾病發(fā)生、發(fā)展和預后的重要指標。β2微球蛋白的檢測方法主要有免疫比濁法、免疫透射比濁法、化學發(fā)光法等，其中酶聯(lián)免疫法因簡便、快速、敏感性高而備受臨床歡迎，而目前可應用于臨床的ELISA試劑盒較少，因此研制一種快捷有效的ELISA檢測方法具有重要意義。 本研究以高純度的人β2微球蛋白作為免疫原，采用常規(guī)免疫和脾內免疫相結合的方法免疫純系BALB/c小鼠，取免疫脾細胞與對數期SP2/0細胞進行融合，間接ELISA法篩選陽性孔。經3~4次克隆化、多次傳代及凍存后復蘇，，共篩選獲得5株穩(wěn)定分泌抗人β2微球蛋白的單克隆抗體雜交瘤細胞株，分別命名為2G7、2H2、3F9、5D10、8E12。經鑒定，5株單抗亞型皆為IgG1類，腹水效價均在10-7-10-9之間，腹水經Protein-A親和層析法純化后進行電泳，結果顯示5株單抗純度良好。間接ELISA法和Wersten blot鑒定單抗特異性，結果表明5株單抗均能與人β2微球蛋白發(fā)生特異結合，而與人血清白蛋白、血紅蛋白、EB病毒基因工程抗原、支原體基因工程抗原無交叉反應，證明5株單抗具有良好的特異性和反應原性。 采用改良過碘酸鈉法分別對5株單抗進行酶標，經抗體配對實驗篩選出一對可用于雙抗體夾心ELISA的配對抗體，初步建立了β2微球蛋白雙抗體夾心定量ELISA檢測方法。經方陣滴定實驗確定了包被抗體最佳包被濃度為3ug/mL，酶標二抗最佳工作濃度為1：4000，該檢測方法線性范圍為1.25～40ng/mL，最低檢出限為1.25ng/mL，批內CV＜8%，批間CV＜12%，準確度良好。用本研究建立的檢測方法檢測35份臨床血清標本β2微球蛋白含量，與Roche公司的β2微球蛋白檢測試劑盒檢測結果相比，相關系數R2=0.9550，兩者具有良好的相關性。 本研究成功制備了抗人β2微球蛋白單克隆抗體雜交瘤細胞株，初步建立了人β2微球蛋白雙抗體夾心定量ELISA檢測方法，為進一步研制快速診斷試劑盒和其他檢測方法奠定了良好的基礎。
[Abstract]:尾 2 microglobulin (尾 2 MG) is an endogenous low molecular weight serum protein produced by the body. Molecular weight of 11.8 KD, a class I molecule of major histocompatibility complex, plays an important role in immune response. With metabolism and degradation of MHC, 尾 2 microglobulin is isolated in serum and urine. The content of 尾 2 microglobulin in cerebrospinal fluid, saliva and other body fluids is low and constant. In recent years, the application of 尾 2 microglobulin in basic medicine and clinical diagnosis has become a research hotspot. The change of 尾 2 microglobulin in serum or urine is an important index to evaluate the function of organ and the occurrence, development and prognosis of some diseases. The main methods of detecting 尾 2 microglobulin are immune turbidimetry and immune transmission turbidimetry. Among the chemiluminescence methods, enzyme-linked immunosorbent assay (Elisa) is very popular in clinic because of its simplicity, rapidity and high sensitivity. However, there are few ELISA kits that can be used in clinic at present, so it is of great significance to develop a rapid and effective method for ELISA detection. In this study, high purity human 尾 2 microglobulin was used as immunogen to immunize pure BALB/c mice with routine and intrasplenic immunizations. Spleen cells were taken to fuse with SP2/0 cells in logarithmic phase. Five monoclonal antibody hybridoma cell lines secreting stable anti-human 尾 2 microglobulin were obtained by indirect ELISA method. After 3 times of cloning, repeated passage and resuscitation after cryopreservation, 5 hybridoma cell lines secreted stably anti-human 尾 2 microglobulin were obtained. The McAbs were identified as IgG1 subtypes and ascites titers ranged from 10-7 to 10-9. The purified ascites were purified by Protein-A affinity chromatography. The results showed that the purity of the McAbs was good. Indirect ELISA and Wersten blot were used to identify the McAbs. The results showed that all of the 5 McAbs could specifically bind to human 尾 2 microglobulin, but had no cross reaction with human serum albumin, hemoglobin, EB virus gene engineering antigen and mycoplasma gene engineering antigen. It was proved that the five McAbs had good specificity and reactivity. The modified sodium periodate method was used to label 5 McAbs respectively. A pair of paired antibodies which could be used for double antibody sandwich ELISA was screened by antibody pairing test. A method for the determination of 尾 2 microglobulin double antibody sandwich quantitative ELISA was established. The optimum concentration of coated antibody was determined to be 3ugr / mL by square array titration, and the optimal working concentration of enzyme labeled antibody was 1: 4000. the linear range of the method was 1.254ng / mL, and the lowest was determined as follows: 1: 4000. The detection limit was 1.25 ng 路mL ~ (-1), within-run CV < 8 and between-batch CV < 12, and the accuracy was good. The 尾 _ 2-microglobulin content in 35 clinical serum samples was detected by the method established in this study. Compared with the 尾 2 microglobulin kit of Roche Company, the correlation coefficient was 0.9550, and there was a good correlation between the two. In this study, hybridoma cell lines with monoclonal antibodies against human 尾 2 microglobulin were successfully prepared, and a sandwich quantitative ELISA method for detection of double antibodies against human 尾 2 microglobulin was established. It lays a good foundation for the further development of rapid diagnostic kit and other detection methods.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R943

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