抗hBLyS多、单克隆抗体的研制及应用
发布时间：2018-08-04 19:20
【摘要】：人B淋巴细胞刺激因子(hBLyS)是1999年发现的肿瘤坏死因子超家族新成员,它与某些免疫疾病的制病机制密切相关,其低量或过量表达均能引起机体的免疫失衡。本研究以大肠杆菌BL21(λDE3)表达重组hBLyS的可溶性蛋白为抗原,分别免疫日本大耳白兔和BALB/c小鼠,制备出抗hBLyS的多克隆和单克隆抗体,并对这两种抗体的一些特性进行鉴定。此外,应用这两种抗体建立hBLyS的双抗夹心ELISA检测方法。 1 抗hBLyS多克隆抗体的制备与鉴定 选取2～3kg的雄性日本大耳白兔,淋巴结注射弗氏佐剂乳化的可溶性hBLyS蛋白,每间隔2周注射一次;第3次免疫后11天,耳动脉采血,间接ELISA检测抗血清的效价为50万倍;颈总动脉放血,制备抗血清,并应用分级饱和硫酸铵盐析法进行纯化,westen-blot分析抗体的特异性。 2 抗hBLyS单克隆抗体的制备与特性鉴定 用hBLyS蛋白免疫BALB/c小鼠,取免疫脾细胞与骨髓瘤SP2/0细胞进行融合,通过5次亚克隆获得1株分泌抗hBLyS单克隆抗体的杂交瘤细胞,,染色体数目在87～101之间:该抗体属于IgG1类蛋白,其腹水效价可达2×10~6通过western-blot鉴定其特异性良好。采用小鼠腹腔注射法大量制备抗体,A蛋白亲和层析法纯化抗体。 3 建立检测hBLyS蛋白含量的ELISA方法 选用2种ELISA方案对hBLyS蛋白含量的测定方法进行研究,方案一用链霉亲和素做间接包被,方案二用生物素-亲和素系统做终反应放大;优化选择2种检测方法的反应条件;方法一的敏感度较低:方法二在hBLyS蛋白浓度为3.2～400 ng/mL范围内,标准曲线的线性关系良好;从而建立了一种可检测hBLyS蛋白浓度为ng级水平的ELISA方法。
[Abstract]:Human B lymphocyte stimulating factor (hBLyS) is a new member of the tumor necrosis factor superfamily discovered in 1999. It is closely related to the pathogenesis of some immune diseases, and its low or excessive expression can cause immune imbalance. In this study, the soluble protein of Escherichia coli BL21 (位 DE3) expressing recombinant hBLyS was used as antigen, Japanese white rabbits and BALB/c mice were immunized, polyclonal and monoclonal antibodies against hBLyS were prepared, and some characteristics of these two antibodies were identified. In addition, Using these two antibodies to establish a double antibody sandwich ELISA detection method for hBLyS. 1 preparation and identification of anti hBLyS polyclonal antibody Male Japanese white rabbits with 2~3kg, Lymph nodes were injected with soluble hBLyS protein emulsified by Freund's adjuvant and injected once every 2 weeks. 11 days after the third immunization, blood was collected from ear artery, the titer of antiserum detected by indirect ELISA was 500000 times. The specificity of the antibody was analyzed by Western blot with the method of fractionated saturated ammonium sulfate saltout. 2 preparation and characterization of monoclonal antibody against hBLyS BALB/c mice were immunized with hBLyS protein. Immune spleen cells were fused with myeloma SP2/0 cells and a hybridoma cell line secreting monoclonal antibody against hBLyS was obtained by five subclones. The chromosome number of the hybridoma cells was between 87101.The antibody belongs to IgG1 protein. Its ascites titer can reach 2 脳 10 ~ (-6). Its specificity is good by western-blot. The antibody was purified by affinity chromatography of antibody A protein by intraperitoneal injection of mice. (3) A ELISA method for the detection of hBLyS protein was established. Two kinds of ELISA schemes were used to determine the content of hBLyS protein. In scheme one, streptavidin was used as indirect coating, and biotin-avidin system was used to amplify the final reaction, and the reaction conditions of two detection methods were optimized. The first method has low sensitivity: the second method has a good linear relationship in the range of 3.2O400 ng/mL of hBLyS protein concentration, thus a ELISA method for detecting hBLyS protein concentration at ng level was established.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
本文编号：2164887
[Abstract]:Human B lymphocyte stimulating factor (hBLyS) is a new member of the tumor necrosis factor superfamily discovered in 1999. It is closely related to the pathogenesis of some immune diseases, and its low or excessive expression can cause immune imbalance. In this study, the soluble protein of Escherichia coli BL21 (位 DE3) expressing recombinant hBLyS was used as antigen, Japanese white rabbits and BALB/c mice were immunized, polyclonal and monoclonal antibodies against hBLyS were prepared, and some characteristics of these two antibodies were identified. In addition, Using these two antibodies to establish a double antibody sandwich ELISA detection method for hBLyS. 1 preparation and identification of anti hBLyS polyclonal antibody Male Japanese white rabbits with 2~3kg, Lymph nodes were injected with soluble hBLyS protein emulsified by Freund's adjuvant and injected once every 2 weeks. 11 days after the third immunization, blood was collected from ear artery, the titer of antiserum detected by indirect ELISA was 500000 times. The specificity of the antibody was analyzed by Western blot with the method of fractionated saturated ammonium sulfate saltout. 2 preparation and characterization of monoclonal antibody against hBLyS BALB/c mice were immunized with hBLyS protein. Immune spleen cells were fused with myeloma SP2/0 cells and a hybridoma cell line secreting monoclonal antibody against hBLyS was obtained by five subclones. The chromosome number of the hybridoma cells was between 87101.The antibody belongs to IgG1 protein. Its ascites titer can reach 2 脳 10 ~ (-6). Its specificity is good by western-blot. The antibody was purified by affinity chromatography of antibody A protein by intraperitoneal injection of mice. (3) A ELISA method for the detection of hBLyS protein was established. Two kinds of ELISA schemes were used to determine the content of hBLyS protein. In scheme one, streptavidin was used as indirect coating, and biotin-avidin system was used to amplify the final reaction, and the reaction conditions of two detection methods were optimized. The first method has low sensitivity: the second method has a good linear relationship in the range of 3.2O400 ng/mL of hBLyS protein concentration, thus a ELISA method for detecting hBLyS protein concentration at ng level was established.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
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