本文關(guān)鍵詞：抗雞IL-4單克隆抗體的研制及其雙抗體夾心ELISA方法的初步建立　出處：《揚(yáng)州大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 雞白細(xì)胞介素4 單克隆抗體 抗體夾心ELISA

【摘要】： IL-4是一種多效性T細(xì)胞源細(xì)胞因子,通過作用于不同類型細(xì)胞來調(diào)節(jié)宿主防御和宿主免疫。IL-4在調(diào)節(jié)T、B細(xì)胞分化及Th2型免疫反應(yīng)的發(fā)展中是必需的。此外,IL-4也是單核細(xì)胞和巨噬細(xì)胞激活的調(diào)節(jié)因子。 已有大量的文獻(xiàn)研究了人、小鼠、大鼠、豬等哺乳動(dòng)物的IL-4基因及其生物學(xué)活性。在家禽中,因缺乏禽類相關(guān)的特異性試劑而阻礙了禽類IL-4的研究,本研究旨在以通過制備雞IL-4特異性單克隆抗體為基礎(chǔ),初步建立雞IL-4雙單抗夾心ELISA方法,為家禽的免疫檢測(cè)、免疫細(xì)胞功能分析和免疫調(diào)節(jié)等方面的研究提供條件。 一、抗雞IL-4單克隆抗體的制備與鑒定 應(yīng)用淋巴細(xì)胞雜交瘤技術(shù),用純化的重組蛋白rGST-ChIL-4免疫8周齡BALB/c小鼠,腹部皮下和腹腔免疫,100μg/只。首免時(shí),抗原與等量弗氏完全佐劑混合乳化經(jīng)腹部皮下免疫;二免時(shí)抗原與等量不完全弗氏佐劑混合乳化經(jīng)腹部皮下免疫;三免時(shí)用抗原通過腹腔直接免疫;間隔為兩周;尾靜脈加強(qiáng)免疫不加佐劑的抗原,100μg/只,3d后,取免疫鼠脾細(xì)胞和骨髓瘤SP2/0-Ag-14細(xì)胞進(jìn)行融合。純化的rHis-ChIL-4作為檢測(cè)抗原,用間接ELISA方法篩選陽(yáng)性克隆。獲得9株穩(wěn)定分泌ChIL-4的單克隆抗體細(xì)胞株,命名為16D8、17D7、18F7、18F12、19F1、20C9、20F2、20G3、6A8,它們的腹水效價(jià)依次為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亞類為IgG2a外,其余均為IgG1。 試驗(yàn)表明,所得單抗只與相應(yīng)的融合蛋白反應(yīng),與其它融合蛋白和對(duì)照細(xì)菌不反應(yīng),顯示良好的特異性。Western-blot試驗(yàn)顯示所得單抗均能與相應(yīng)的融合蛋白發(fā)生反應(yīng),出現(xiàn)特異性條帶。為雞體內(nèi)IL-4水平的檢測(cè),作為某些疾病的檢測(cè)指標(biāo),用于疾病的預(yù)防和控制,進(jìn)行細(xì)胞免疫機(jī)理及細(xì)胞因子間相互作用方面的研究提供了有用的材料。 二、雞IL-4雙單抗夾心ELISA方法的初步建立 通過對(duì)6株單抗抗體飽和度測(cè)定和相加指數(shù)測(cè)定進(jìn)行初步篩選,采用20C9單抗以濃度為10μg/ml作為包被抗體,酶標(biāo)抗體bio-16D8作為檢測(cè)抗體建立抗體夾心ELISA方法,并摸索封閉液、稀釋液、抗原最佳反應(yīng)時(shí)間、酶標(biāo)抗體最佳工作濃度、反應(yīng)時(shí)間、HRP-streptavidin最佳工作濃度、反應(yīng)時(shí)間、底物最佳反應(yīng)時(shí)間等條件,對(duì)比試驗(yàn)最終選擇含10%小牛血清的磷酸鹽緩沖液作為封閉液,含4%PEG、0.05%Tween-20的磷酸鹽緩沖液作為稀釋液,抗原反應(yīng)時(shí)間為2h,酶標(biāo)抗體最佳工作濃度為2μg/ml、最適反應(yīng)時(shí)間為30min,HRP-streptavidin最佳工作濃度為1:2,000、最佳反應(yīng)時(shí)間為30min,底物最佳反應(yīng)時(shí)間為5min。此方法可檢出的ChIL-4為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).
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前2條

1 蔡文博;李行;高彩霞;武永淑;楊柳;張偉;韓凌霞;;Th1和Th2型細(xì)胞因子在不同MHC-B單倍型雞群中的轉(zhuǎn)錄分析[J];免疫學(xué)雜志;2011年12期

2 蔡文博;韓凌霞;;Th1、Th2型細(xì)胞因子在雞馬立克氏病中的研究進(jìn)展[J];細(xì)胞與分子免疫學(xué)雜志;2011年07期

相關(guān)碩士學(xué)位論文 前1條

1 蔡文博;Th1和Th2型細(xì)胞因子在不同MHC-B單倍型雞群中的表達(dá)分析[D];中國(guó)農(nóng)業(yè)科學(xué)院;2011年

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本文編號(hào)：1367620