丙氨酸氨基转移酶单克隆抗体的制备及鉴定
发布时间：2019-06-25 21:28
【摘要】： 研究背景:丙氨酸氨基转移酶是人血清中的一种重要的催化酶,在蛋白质分解代谢中起氨基转移作用。它主要存在于肝细胞浆内,肝细胞损伤时,大量的ALT释放入血,所以丙氨酸氨基转移酶是反映肝损伤的敏感的指标。为减少输血传播疾病的发生,我国卫生部规定,血清ALT检验是献血员健康体检必查项目之一,而我国每年由于ALT不合格造成的采血后血液报废率达6-8%。面对现在街头无偿献血者因血清ALT高造成大量不必要的献血和血液浪费问题,非常有必要建立一种快速灵敏、准确方便的ALT检测方法。目前街头献血者ALT的筛查均采用速率法和化学干粉法。但这两种方法存在的主要问题是反应时间长,需要特殊设备,受环境因素影响大。免疫金标法具有操作简便、快速、特异性强,不需要特殊设备,易观察结果等优点,因此我们探索ALT免疫金标快速检测试剂的研制,而高特异性抗ALT单克隆抗体的制备是该试剂研制的关键步骤。 目的:获得分泌高特异性抗ALT单克隆抗体的杂交瘤细胞株;制备针对ALT的单克隆抗体并对其特性进行鉴定。 方法:用含50%福氏佐剂的猪ALT纯品(20μg)腹部皮下免疫Balb/C小鼠,共4次,免疫间隔为2周。间接ELISA法测抗体效价。取效价满意鼠的脾细胞与Sp2/0骨髓瘤细胞进行融合,用含HAT和HT的培养液选择性培养,用高ALT人血清作检测原,采用间接ELISA法测定培养上清中抗体分泌情况,阳性孔经三次有限稀释法克隆化,得到的阳性杂交瘤细胞株腹腔接种于预注射降植烷的Balb/C小鼠,收集腹水,用正辛酸-硫酸铵沉淀法进行纯化。 采用间接ELISA法测定腹水中效价和亲和力;秋水仙素阻断法进行杂交瘤细胞染色体分析;用Southern Biotech公司的单克隆抗体亚类鉴定试剂鉴定单抗亚类;SDS-PAGE电泳测定单抗的分子量;用Western blotting分析单克隆抗体的特异性;将纯化后单抗用辣根过氧化物酶标记,建立双抗体夹心ELISA检测人血清中的ALT的方法。 结果:采用传统的细胞融合技术得到1株分泌抗人ALT单克隆抗体的杂交瘤细胞株,其染色体数目为95～106。其分泌的抗体为IgG1,轻链为κ型。上清和腹水中抗体效价分别为10~(-4)和10~(-6)。SDS-PAGE显示该抗体在55KD和25KD Western blotting结果显示该株McAb可与人血清中的ALT特异地结合。人血清标本和ALT质控品的双抗体夹心ELISA结果和生化分析仪结果基本一致。结论:本研究获得了一株能稳定分泌ALT单克隆抗体特异性的杂交瘤细胞株,该株单抗有很好的亲和力,能特异性地识别人血清中的ALT,而与血清中的其它蛋白无交叉反应。本研究为建立ALT的免疫金标检测方法提供了很有价值的试验工具。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.11
本文编号：2506025
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.11
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