抗鸡IL-4单克隆抗体的研制及其双抗体夹心ELISA方法的初步建立

发布时间：2018-01-02 04:14

本文关键词：抗鸡IL-4单克隆抗体的研制及其双抗体夹心ELISA方法的初步建立　出处：《扬州大学》2009年硕士论文　论文类型：学位论文

【摘要】： IL-4是一种多效性T细胞源细胞因子,通过作用于不同类型细胞来调节宿主防御和宿主免疫。IL-4在调节T、B细胞分化及Th2型免疫反应的发展中是必需的。此外,IL-4也是单核细胞和巨噬细胞激活的调节因子。已有大量的文献研究了人、小鼠、大鼠、猪等哺乳动物的IL-4基因及其生物学活性。在家禽中,因缺乏禽类相关的特异性试剂而阻碍了禽类IL-4的研究,本研究旨在以通过制备鸡IL-4特异性单克隆抗体为基础,初步建立鸡IL-4双单抗夹心ELISA方法,为家禽的免疫检测、免疫细胞功能分析和免疫调节等方面的研究提供条件。一、抗鸡IL-4单克隆抗体的制备与鉴定应用淋巴细胞杂交瘤技术,用纯化的重组蛋白rGST-ChIL-4免疫8周龄BALB/c小鼠,腹部皮下和腹腔免疫,100μg/只。首免时,抗原与等量弗氏完全佐剂混合乳化经腹部皮下免疫;二免时抗原与等量不完全弗氏佐剂混合乳化经腹部皮下免疫;三免时用抗原通过腹腔直接免疫;间隔为两周;尾静脉加强免疫不加佐剂的抗原,100μg/只,3d后,取免疫鼠脾细胞和骨髓瘤SP2/0-Ag-14细胞进行融合。纯化的rHis-ChIL-4作为检测抗原,用间接ELISA方法筛选阳性克隆。获得9株稳定分泌ChIL-4的单克隆抗体细胞株,命名为16D8、17D7、18F7、18F12、19F1、20C9、20F2、20G3、6A8,它们的腹水效价依次为1,280,000、1,280,000、640,000、640,000、640,000、640,000、640,000、640,000、160,000、160,000,除16D8和20F2 McAb亚类为IgG2a外,其余均为IgG1。试验表明,所得单抗只与相应的融合蛋白反应,与其它融合蛋白和对照细菌不反应,显示良好的特异性。Western-blot试验显示所得单抗均能与相应的融合蛋白发生反应,出现特异性条带。为鸡体内IL-4水平的检测,作为某些疾病的检测指标,用于疾病的预防和控制,进行细胞免疫机理及细胞因子间相互作用方面的研究提供了有用的材料。二、鸡IL-4双单抗夹心ELISA方法的初步建立通过对6株单抗抗体饱和度测定和相加指数测定进行初步筛选,采用20C9单抗以浓度为10μg/ml作为包被抗体,酶标抗体bio-16D8作为检测抗体建立抗体夹心ELISA方法,并摸索封闭液、稀释液、抗原最佳反应时间、酶标抗体最佳工作浓度、反应时间、HRP-streptavidin最佳工作浓度、反应时间、底物最佳反应时间等条件,对比试验最终选择含10%小牛血清的磷酸盐缓冲液作为封闭液,含4%PEG、0.05%Tween-20的磷酸盐缓冲液作为稀释液,抗原反应时间为2h,酶标抗体最佳工作浓度为2μg/ml、最适反应时间为30min,HRP-streptavidin最佳工作浓度为1:2,000、最佳反应时间为30min,底物最佳反应时间为5min。此方法可检出的ChIL-4为0.039μg/ml。
[Abstract]:IL-4 is a multipotent T cell-derived cytokine that regulates T by acting on different types of cells to regulate host defense and host immune. IL-4. B cell differentiation and the development of Th2 type immune response are necessary. In addition, IL-4 is also a regulatory factor for the activation of monocytes and macrophages. The IL-4 gene and its biological activity in human, mouse, rat, pig and other mammals have been studied in a large number of literatures. The research of avian IL-4 is hindered by the lack of specific reagents related to poultry. This study is based on the preparation of specific monoclonal antibodies against chicken IL-4. A sandwich ELISA method for chicken IL-4 double monoclonal antibody was established, which provided conditions for the study of immune detection, analysis of immune cell function and immunomodulation of poultry. Preparation and Identification of Monoclonal Antibodies against Chicken IL-4 Lymphocyte hybridoma technique was used to immunize 8-week-old BALB/c mice with purified recombinant protein rGST-ChIL-4. The mice were immunized subcutaneously and intraperitoneally with 100 渭 g / mouse for the first time. Antigens and equivalent Freund's complete adjuvant were mixed emulsified and immunized subcutaneously through abdomen. Mixed emulsification of antigen and equal amount of incomplete Freund's adjuvant was subcutaneously immunized through abdomen. Three immunizations were directly immunized with antigens through abdominal cavity. The interval is two weeks; The vena caudalis was immunized with 100 渭 g of antigen without adjuvant only 3 days later. Spleen cells of immunized mice and SP2/0-Ag-14 cells of myeloma were fused and purified rHis-ChIL-4 was used as antigen. Nine monoclonal antibody cell lines stably secreting ChIL-4 were obtained by indirect ELISA screening. The ascites titer of 20C9 / 20F2 / 20G3 / 6A8 is 1 / 280 / 000 / kg / kg / kg / kg / kg / kg / kg / kg / kg / kg / kg / kg, and their ascites titer is 1 / 280 / 000 / kg / kg / kg / kg / kg / kg / kg / kg, respectively. The subclasses of 16D8 and 20F2 were IgG2a, except 16D8 and 20F2 McAb subclasses. The rest were IgG1. The results showed that the McAbs reacted only with the corresponding fusion protein, but not with other fusion proteins and control bacteria. Western-blot test showed that the McAbs could react with the corresponding fusion protein, and there were specific bands, which was the detection of IL-4 level in chicken. As the detection index of some diseases, it provides useful materials for the prevention and control of diseases, the study of cellular immune mechanism and the interaction between cytokines. Second, the establishment of a sandwich ELISA method for chicken IL-4 double monoclonal antibody The antibody saturation and additive index of 6 strains of McAbs were preliminarily screened. The concentration of 20C9 McAb was 10 渭 g / ml as the coating antibody. Enzyme labeled antibody (bio-16D8) was used as an antibody detection method to establish an antibody sandwich ELISA method. The best reaction time of antigens, the best concentration of enzyme labeled antibody, and the reaction time were explored in the blocking solution, diluent solution, antigen and enzyme labeled antibody. The optimal working concentration of HRP-streptavidin, reaction time and substrate reaction time were optimized. The phosphate buffer containing 10% calf serum was selected as the blocking solution. The phosphate buffer containing 4PEG0. 05 and Tween-20 was used as diluent, the antigenic reaction time was 2 h, and the optimal working concentration of enzyme labeled antibody was 2 渭 g / ml. The optimum reaction time was 30 min HRP-streptavidin was 1: 2 000, and the optimum reaction time was 30 min. The optimum reaction time of the substrate was 5 min. The ChIL-4 detected by this method was 0.039 渭 g 路ml ~ (-1) 路ml ~ (-1).
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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