本文關鍵詞：高遷移率族蛋白B1單克隆抗體的制備及其在競爭ELISA中的應用　出處：《天津醫(yī)科大學》2007年碩士論文　論文類型：學位論文

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【摘要】： 目的制備HMGB1單克隆抗體并建立HMGB1的競爭ELISA測定方法。 方法用bis(sulfosuccinimidyl)suberate制備HMGB1-BSA偶聯(lián)物(HMGB1-BSA)和HRP標記的HMGB1(HMGB1-HRP)；以完全佐劑乳化的HMGB1-BSA免疫Balb／c小鼠；用50％PEG使小鼠脾細胞與Sp2／0細胞融合；用有限稀釋法克隆雜交瘤細胞；用DE52離子交換色層析柱純化單克隆抗體；用靜脈注射鏈尿菌素法建立大鼠糖尿病模型。 結果在120個孔內，發(fā)現(xiàn)17個孔內有分泌HMGB1單克隆抗體的雜交瘤細胞生長。對其中A6-Ⅲ孔內雜交瘤細胞用有限稀釋法克隆化，共獲得7株分泌HMGB1特異性抗體的雜交瘤細胞株。用其中一株雜交瘤(HMGB1-C1株)制備了純化HMGB1單克隆抗體。利用HMGB1-BSA和HMGB1-HRP建立的競爭ELISA的測定范圍為1～320ng／mL，回收率為97～106％，變異系數(shù)為2.6～7.3％。用該競爭ELISA測定正常和糖尿病大鼠血清HMGB1水平發(fā)現(xiàn)糖尿病大鼠血清HMGB1水平顯著高于正常大鼠。 結論以HMGB1單克隆抗體建立的競爭ELISA有望成為一種具有靈敏度高、特異性強，，重復性好，操作簡便和快速的測定方法。
[Abstract]:Objective to prepare HMGB1 monoclonal antibody and establish a competitive ELISA assay for HMGB1. Methods the HMGB1-BSA coupling compound HMGB1-BSA was prepared by bis(sulfosuccinimidyl)suberate. And HRP labeled HMGB1, HMGB1-HRPU; Balb/c mice were immunized with complete adjuvant emulsified HMGB1-BSA. Mouse spleen cells were fused with Sp2/0 cells with 50PEG. The hybridoma cells were cloned by limited dilution method. Monoclonal antibody was purified by DE52 ion exchange chromatography. The diabetic model of rats was established by intravenous streptozotocin. Results the hybridoma cells secreting monoclonal antibodies to HMGB1 were found in 17 of 120 holes, and the hybridoma cells in hole A6- 鈪

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