【作者】 谭璐；

【导师】 吕永宁； 陈东生；

【作者基本信息】 华中科技大学 ， 药剂学， 2009， 硕士

【摘要】 目的:研究环孢素A完全抗原的合成及单克隆抗体制备的方法。方法:室温搅拌条件下,在脱水剂1-乙基-(3-二甲丙胺基)碳二亚胺盐酸盐(EDCI)的作用下,将环孢素A与4-苯甲酰苯甲酸的共轭物(CsA-BBa)与聚L-赖氨酸(PL)按质量比为3.4:1的比例进行反应。反应产物用截留量1万的透析膜透析分离、纯化。在190-300nm范围内对合成抗原进行紫外图谱扫描鉴定;用5μg/ml的抗原包被酶标板,酶联免疫吸附法(ELISA)进行抗原特异性鉴定。用完全抗原免疫七周龄雌性BALB/C小鼠, ELISA法检测免疫小鼠血清抗体效价。在50%聚乙二醇(PEG)的作用下,将高效价小鼠脾细胞与骨髓瘤细胞进行融合(脾细胞:骨髓瘤细胞=10:1)并接种于24 h前准备的饲养层细胞培养板中,HAT培养液对融合细胞进行选择性培养,用ELISA法对杂交瘤细胞进行筛选、鉴定。结果:环孢素A完全抗原(CsA-BBa-PL)紫外吸收峰峰位209.5nm,较合成原料物质CsA-BBa(212nm)及PL(203nm)发生了明显迁移,证实了产物的生成;环孢素A完全抗原ELISA特异性检测结果与阴性对照的比值为23.22。免疫BALB/C小鼠血清效价值为:3.35,3.43,3.37,3.31,3.38,空白对照、阴性对照、阳性对照值依次为0.080、0.42、3.43。免疫小鼠血清效价测定结果明显大于空白对照及阴性对照值,并与阳性对照值接近。所筛选的杂交瘤细胞能够稳定分泌环孢素A单克隆抗体。结论:该实验方法能够有效的合成环孢素A完全抗原;合成抗原能够刺激BALB/C小鼠机体产生免疫应答反应并产生相应环孢素A抗体;实验所采用杂交瘤技术能够成功的制备环孢素A单克隆抗体。更多还原

【Abstract】 OBJECTIVE: To study the methodology of immunogen synthesis and the monoclonal antibody preparation of Cyclosporine A.METHODS: With the stirring at room temperature and dehydrolyzing agent 1-ethyl-3-(3’-dimethylaminopropyl) carbodiimide (EDCI ), the antigen of cyclosporine A(CsA-BBa-PL) was synthesized through the reaction between the conjugate of cyclosporine A and 4-benzoulbenzoic (CsA-BBa) and poly-L-lysine(PL), the weight proportion was 3.4 to 1. The products were isolated and purified by dialysis, the specification of the dialysis-membrane was 10,000. It was identified by UV spectra within 190 to 300 nm. The specificity of the antigen of CsA was identified by enzyme-linked immunosorbent assay (ELISA), the concentration of antigen used for the identification was 5μg/ml. The female BALB/C mice, seven weeks old, were immunized with the antigen of cyclosporine A, the blood serum potencies were measured by indirect enzyme-linked immunosorbent assay. With the help of fusogenic agent polyethylene glycol (PEG), the concentration of which is 50%, the spleen cells, which had higher blood serum potencies, were fused with SP2/0 myeloma cells (the ratio is ten to one). Then the fused cells were cultured in the cell culture plates which had owned feeder layer cells for 24 hours. The selected solution HAT was used to culture the fusion cells so as to select hybridoma cells. The hybridoma cells was selected and identified by ELISA.RESULTS: The UV spectra peaks of CsA-BBa and PL and the antigen of CsA were 212nm, 203nm, 209.5nm respectively, compared to the materials used to synthesize antigen of cyclosporine A, the UV spectra of the antigen changed obviously, which proved that the conjugate was formed through the reaction. The positive to negative ratio of the specificity identification was 23.22. The blood serum potencies of the immune mice were 3.35, 3.43, 3.37, 3.31, and 3.38 respectively. The blank control and negative control and positive control were 0.080, 0.42 and 3.43 respectively. The potencies of the mice were obviously higher than the blank control and negative control, which were close to the positive control. The selected hybridoma cells were able to secrete monoclonal antibodies of cyclosporine A stably. CONCLUSIONS: The antigen of cyclosporine A was able to synthetized by this reaction. The antigen was able to induce the immunological reaction of mice and made the body form the antibodies accordingly. The hybridoma technique was able to prepare monoclonal antibody of Cyclosporine A successfully.更多还原