抗人β2微球蛋白单克隆抗体的制备及ELISA检测方法的初步建立

发布时间：2018-03-26 03:40

本文选题：β2微球蛋白　切入点：单克隆抗体　出处：《郑州大学》2014年硕士论文

【摘要】：β2微球蛋白（β2-microglobulin，β2-MG）是由机体产生的一种内源性低分子量血清蛋白质，分子量11.8KD，是细胞膜上完整主要组织相容性复合物（majorhistocompatibility complex，MHC）Ⅰ类分子的轻链（β链），在免疫应答中起重要作用。随着代谢和MHC的降解，β2微球蛋白分离后以游离形式存在于血清、尿液、脑脊液、唾液等体液中，含量较低且恒定。近年来，β2微球蛋白在基础医学及临床诊断上的应用已成为研究热点，检测血清或尿液中β2微球蛋白含量的变化成为评价相关脏器功能和某些疾病发生、发展和预后的重要指标。β2微球蛋白的检测方法主要有免疫比浊法、免疫透射比浊法、化学发光法等，其中酶联免疫法因简便、快速、敏感性高而备受临床欢迎，而目前可应用于临床的ELISA试剂盒较少，因此研制一种快捷有效的ELISA检测方法具有重要意义。本研究以高纯度的人β2微球蛋白作为免疫原，采用常规免疫和脾内免疫相结合的方法免疫纯系BALB/c小鼠，取免疫脾细胞与对数期SP2/0细胞进行融合，间接ELISA法筛选阳性孔。经3~4次克隆化、多次传代及冻存后复苏，，共筛选获得5株稳定分泌抗人β2微球蛋白的单克隆抗体杂交瘤细胞株，分别命名为2G7、2H2、3F9、5D10、8E12。经鉴定，5株单抗亚型皆为IgG1类，腹水效价均在10-7-10-9之间，腹水经Protein-A亲和层析法纯化后进行电泳，结果显示5株单抗纯度良好。间接ELISA法和Wersten blot鉴定单抗特异性，结果表明5株单抗均能与人β2微球蛋白发生特异结合，而与人血清白蛋白、血红蛋白、EB病毒基因工程抗原、支原体基因工程抗原无交叉反应，证明5株单抗具有良好的特异性和反应原性。采用改良过碘酸钠法分别对5株单抗进行酶标，经抗体配对实验筛选出一对可用于双抗体夹心ELISA的配对抗体，初步建立了β2微球蛋白双抗体夹心定量ELISA检测方法。经方阵滴定实验确定了包被抗体最佳包被浓度为3ug/mL，酶标二抗最佳工作浓度为1：4000，该检测方法线性范围为1.25～40ng/mL，最低检出限为1.25ng/mL，批内CV＜8%，批间CV＜12%，准确度良好。用本研究建立的检测方法检测35份临床血清标本β2微球蛋白含量，与Roche公司的β2微球蛋白检测试剂盒检测结果相比，相关系数R2=0.9550，两者具有良好的相关性。本研究成功制备了抗人β2微球蛋白单克隆抗体杂交瘤细胞株，初步建立了人β2微球蛋白双抗体夹心定量ELISA检测方法，为进一步研制快速诊断试剂盒和其他检测方法奠定了良好的基础。
[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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

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