【摘要】： 研究背景:丙氨酸氨基轉(zhuǎn)移酶是人血清中的一種重要的催化酶,在蛋白質(zhì)分解代謝中起氨基轉(zhuǎn)移作用。它主要存在于肝細(xì)胞漿內(nèi),肝細(xì)胞損傷時,大量的ALT釋放入血,所以丙氨酸氨基轉(zhuǎn)移酶是反映肝損傷的敏感的指標(biāo)。為減少輸血傳播疾病的發(fā)生,我國衛(wèi)生部規(guī)定,血清ALT檢驗是獻(xiàn)血員健康體檢必查項目之一,而我國每年由于ALT不合格造成的采血后血液報廢率達(dá)6-8%。面對現(xiàn)在街頭無償獻(xiàn)血者因血清ALT高造成大量不必要的獻(xiàn)血和血液浪費問題,非常有必要建立一種快速靈敏、準(zhǔn)確方便的ALT檢測方法。目前街頭獻(xiàn)血者ALT的篩查均采用速率法和化學(xué)干粉法。但這兩種方法存在的主要問題是反應(yīng)時間長,需要特殊設(shè)備,受環(huán)境因素影響大。免疫金標(biāo)法具有操作簡便、快速、特異性強(qiáng),不需要特殊設(shè)備,易觀察結(jié)果等優(yōu)點,因此我們探索ALT免疫金標(biāo)快速檢測試劑的研制,而高特異性抗ALT單克隆抗體的制備是該試劑研制的關(guān)鍵步驟。 目的:獲得分泌高特異性抗ALT單克隆抗體的雜交瘤細(xì)胞株;制備針對ALT的單克隆抗體并對其特性進(jìn)行鑒定。 方法:用含50%福氏佐劑的豬ALT純品(20μg)腹部皮下免疫Balb/C小鼠,共4次,免疫間隔為2周。間接ELISA法測抗體效價。取效價滿意鼠的脾細(xì)胞與Sp2/0骨髓瘤細(xì)胞進(jìn)行融合,用含HAT和HT的培養(yǎng)液選擇性培養(yǎng),用高ALT人血清作檢測原,采用間接ELISA法測定培養(yǎng)上清中抗體分泌情況,陽性孔經(jīng)三次有限稀釋法克隆化,得到的陽性雜交瘤細(xì)胞株腹腔接種于預(yù)注射降植烷的Balb/C小鼠,收集腹水,用正辛酸-硫酸銨沉淀法進(jìn)行純化。 采用間接ELISA法測定腹水中效價和親和力;秋水仙素阻斷法進(jìn)行雜交瘤細(xì)胞染色體分析;用Southern Biotech公司的單克隆抗體亞類鑒定試劑鑒定單抗亞類;SDS-PAGE電泳測定單抗的分子量;用Western blotting分析單克隆抗體的特異性;將純化后單抗用辣根過氧化物酶標(biāo)記,建立雙抗體夾心ELISA檢測人血清中的ALT的方法。 結(jié)果:采用傳統(tǒng)的細(xì)胞融合技術(shù)得到1株分泌抗人ALT單克隆抗體的雜交瘤細(xì)胞株,其染色體數(shù)目為95～106。其分泌的抗體為IgG1,輕鏈為κ型。上清和腹水中抗體效價分別為10~(-4)和10~(-6)。SDS-PAGE顯示該抗體在55KD和25KD Western blotting結(jié)果顯示該株McAb可與人血清中的ALT特異地結(jié)合。人血清標(biāo)本和ALT質(zhì)控品的雙抗體夾心ELISA結(jié)果和生化分析儀結(jié)果基本一致。結(jié)論:本研究獲得了一株能穩(wěn)定分泌ALT單克隆抗體特異性的雜交瘤細(xì)胞株,該株單抗有很好的親和力,能特異性地識別人血清中的ALT,而與血清中的其它蛋白無交叉反應(yīng)。本研究為建立ALT的免疫金標(biāo)檢測方法提供了很有價值的試驗工具。
[Abstract]:Background: alanine aminotransferase is an important catalytic enzyme in human serum and plays an amino transfer role in protein catabolism. It mainly exists in the cytoplasm of hepatocytes. When hepatocytes are injured, a large amount of ALT is released into blood, so alanine aminotransferase is a sensitive index to reflect liver injury. In order to reduce the occurrence of transfusion-borne diseases, the Ministry of Health of our country stipulates that serum ALT test is one of the necessary items for health examination of blood donors, and the rate of post-collection blood scrapping caused by unqualified ALT in China is 6%. In the face of a large number of unnecessary blood donation and blood waste caused by high serum ALT in street unpaid blood donors, it is necessary to establish a rapid, sensitive, accurate and convenient method for ALT detection. At present, ALT screening of street blood donors is carried out by speed method and chemical dry powder method. However, the main problems of these two methods are long reaction time, need special equipment, and are greatly affected by environmental factors. Immunogold method has the advantages of simple operation, rapidity, strong specificity, no need of special equipment, easy to observe results and so on. Therefore, we explore the development of ALT immunogold standard rapid detection reagent, and the preparation of high specific anti-ALT monoclonal antibody is the key step in the development of this reagent. Aim: to obtain hybridoma cell line secreting highly specific anti-ALT monoclonal antibody, prepare monoclonal antibody against ALT and identify its characteristics. Methods: Balb/C mice were immunized subcutaneously with pig ALT (20 渭 g) containing 50% Freund's adjuvant for 4 times at an interval of 2 weeks. The antibody titer was measured by indirect ELISA. The spleen cells of mice with satisfactory titer were fused with Sp2/0 myeloma cells, selectively cultured in the medium containing HAT and HT, detected by high ALT human serum, and the antibody secretion in the culture medium was determined by indirect ELISA method. The positive hybridoma cell lines were cloned by three times of finite dilution method. The positive hybridoma cell lines were inoculated intraperitoneally in Balb/C mice preinjected with phytane, and the ascitic fluid was collected. Purification was carried out by n-octanic acid-ammonium sulfate precipitation method. Indirect ELISA method was used to determine the titer and affinity of ascitic fluid; colchicine blocking method was used to analyze the chromosomes of hybridoma cells; Southern Biotech monoclonal antibody subclass identification reagent was used to identify monoclonal antibody subclass; SDS-PAGE electrophoresis was used to determine the molecular weight of monoclonal antibody; Western blotting was used to analyze the specificity of monoclonal antibody; and the purified monoclonal antibody was labeled with horseradish peroxide to establish a double antibody sandwich ELISA method for the detection of ALT in human serum. Results: a hybridoma cell line secreting monoclonal antibody against human ALT was obtained by traditional cell fusion technique. The chromosome number of hybridoma cell line was 95 脳 106. The antibody secreted by IgG1, was type K of light chain IgG1,. The antibody titers in upper serum and ascitic fluid were 10 ~ (- 4) and 10 ~ (- 6), respectively. SDS-PAGE showed that the antibody could bind specifically to ALT in human serum by 55KD and 25KD Western blotting. The results of double antibody sandwich ELISA in human serum samples and ALT quality control products were basically consistent with those of biochemical analyzer. Conclusion: a hybridoma cell line which can stably secrete ALT monoclonal antibody was obtained. The monoclonal antibody has good affinity and can specifically recognize ALT, in human serum without cross reaction with other proteins in serum. This study provides a valuable test tool for the establishment of immune gold standard detection method for ALT.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392.11

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