小鼠黑色素瘤抗独特型抗体的制备及生物活性研究

发布时间：2017-12-31 18:35

本文关键词：小鼠黑色素瘤抗独特型抗体的制备及生物活性研究　出处：《中国协和医科大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的：制备抗小鼠B16黑色素瘤的抗独特型抗体(Ab2)并进行体外实验和鉴定，探讨通过免疫效应治疗或者预防小鼠B16黑色素瘤的可能性。方法：分别用gp75多肽和B16细胞作为抗原免疫BALB／c小鼠，通过杂交瘤技术制备抗gp75多肽的单克隆抗体(Ab1)和抗B16细胞的单克隆抗体(Ab1)，用ELISA、Western Blot、免疫组织化学、免疫细胞化学、细胞免疫荧光染色等方法研究Ab1的特性。Ab1经胃蛋白酶水解制备F(ab')_2片断，F(ab')_2片断偶联载体蛋白KLH后作为抗原免疫BALB／c小鼠，用ELISA方法研究小鼠免疫血清中Ab2的特性。结果：获得6株能持续、稳定分泌抗gp75多肽单克隆抗体(Ab1)的杂交瘤细胞株，均为IgG1亚类，其中McAb D分泌稳定、特异性强、效价高；获得6株能持续、稳定分泌抗B16细胞单克隆抗体(Ab1)的杂交瘤细胞株，均为IgG2a亚类，其中McAb 4分泌稳定、特异性强、效价高。Western Blot结果显示McAb D识别约75KD的抗原，McAb 4识别约95 KD的抗原。免疫组织化学等结果表明McAb D和McAb 4所识别的抗原均定位于细胞膜和细胞浆。检测Ab1的F(ab')_2片断免疫血清，结果血清中检测到Ab2和Ab3，Ab2与Ag竞争结合Ab1。结论： Ab1对其抗原有特异亲和性，，在小鼠体内能诱发独特型免疫网络级联反应，为进一步研究抗独特型抗体奠定了基础。
[Abstract]:Objective: To prepare anti-idiotypic antibody (Ab2) against mouse B16 melanoma and to investigate the possibility of immunotherapy or prevention of B16 melanoma in vitro. Methods: Gp75 polypeptides and B16 cells were used as antigens to immunize BALB/c mice. Hybridoma technique was used to prepare monoclonal antibody against gp75 polypeptide (Anab1) and anti-B16 cell line (B16 cell line). Western Blot was used to prepare the monoclonal antibody against B16 cells. Immunohistochemistry, immunocytochemistry and immunofluorescence staining were used to study the characteristics of Ab1. Ab1 was hydrolyzed by pepsin to prepare the fragment of Ab1. The BALB/c mice were immunized with the carrier protein (KLH) from the tip2 fragment. The characteristics of Ab2 in the immune serum of the mice were studied by ELISA method. Results: Six hybridoma cell lines, all of which were subclasses of IgG1, were obtained, which were stable and secreted monoclonal antibody against gp75 polypeptide. Among them, McAb D secreted stably and specifically. High titer; Six hybridoma cell lines, all of which secreted monoclonal antibody against B16 cells, were obtained, all of which were subclasses of IgG2a. Among them, McAb 4 secreted stably and specifically. The results of high titer. Western Blot showed that McAb D recognized about 75KD antigen. McAb 4 recognizes about 95 KD antigens. The results of immunohistochemistry indicate that the antigens recognized by McAb D and McAb 4 are located in cell membrane and cytoplasm. Ab')_2 fragment immunized serum. Results Ab2 and Ab3 + Ab2 combined with Ag were detected in serum. Conclusion: Ab1 has specific affinity to its antigen, which can induce the cascade reaction of idiotypic immune network in mice, which lays a foundation for the further study of anti-idiotypic antibody.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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