《氯睾酮单克隆抗体的制备及鉴定》-毕业论文（设计）.docVIP

精品 PAGE 精品 河　南　科　技　学　院 2011届本科毕业论文（设计） 论文题目：氯睾酮单克隆抗体的制备及鉴定 学生姓名： 所在院（系）：动物科学学院 所学专业： 动物医学 导师姓名： 完成时间： 2011年6月 摘 要 用混合酸酐法将BSA和OVA分别和氯睾酮 (Clostebol，CLO) 偶联作为免疫原和包被原，并经紫外扫描 (UV Scan) 、红外扫描(IR Scan)鉴定。用合成的CLO-BSA免疫Balb/C小鼠，经过5次免疫后，用间接ELISA和阻断ELISA选择细胞融合备用鼠，选择高效价、敏感的小鼠进行抗原超强免疫；取其脾细胞应用杂交瘤技术与骨髓瘤细胞建立分泌CLO单克隆抗体的杂交瘤细胞株；用体内诱生腹水法制备CLO mAb，对CLO mAb的效价、敏感性和特异性等免疫学特性进行鉴定。紫外扫描和红外扫描鉴定表明CLO-BSA人工抗原偶联成功；免疫的3只小鼠血清抗体效价均达到104；其中2号小鼠血清CLO抑制效价较高且IC50最低，达48.59 μg/L，融合后筛选出Z3G12B3和Z2H8C10两株敏感特异的杂交瘤细胞，其两株细胞培养上清液效价1:512，腹水效价为1:2.56×105，Z3G12B3株对CLO的IC50为2.47 μg/L，与睾酮、群勃龙有小于0.03%的交叉反应性，与其他抑制物无交叉反应性。该试验获得了高效价、敏感、特异的抗 CLO mAb，为CLO残留ELISA检测试剂盒和试纸条的建立奠定了坚实的基础。 关键词：氯睾酮，半抗原，人工抗原，单克隆抗体，杂交瘤，免疫学特性 Preparation and Identification of Monoclonal Antibody Against Clostebol Abstract The Clostebol (CLO) was conjugated to carrier protein BSA and OVA by methods of mixed acid anhydride to obtain the artificial and coating antigen. The ultraviolet scanning and infrared scanning were used to identify clostebol artificial antigen. Balb/C mice were immunized with CLO-BSA. The titer of polyclonal antibody (pAb) was detected by indirect ELISA and blocking ELISA after three times immunization. The high titer, sensitive and specific mouse was selected for cell fusing. The hybridoma lines that secrete CLO mAb were established with using monoclonal antibody hybridoma technology and the immunological characteristics such as titer, sensitivity and specificity of the mAb were characterized. The ultraviolet scanning and infrared scanning results showed that CLO artificial antigen was synthesized successfully. Three Balb/C mice indirect ELISA titer against CLO were all above 104 and the IC50 of No. 2 mice was 48.59 μg/L. Two hybridoma cell lines of Z3G12B3 and Z2H8C10 were screened for specificity to CLO after cell fusing, the indirect ELISA titer of the mAb were 1:512 in supernatant, 1:2.56×105 in ascites, the mAb of Z3G12B3 showed good sensitivity with an IC50 of 2.47μg/L to CLO,