促肾上腺皮质激素单克隆抗体的筛选和鉴定  

Preparation and characterisation of monoclonal antibodies to adrenocorticotropin

作　　者：汪保安[1] 汪寅章[1] 夏风城[1] 陆思珍[1] 黄兆坚[1] 郭爱岩[1] 马芳玲[1] 张育青[1] 

机构地区：[1]解放军总医院内分泌科,北京100853

出　　处：《免疫学杂志》2001年第5期388-390,共3页Immunological Journal

摘　　要：目的制备促肾上腺皮质激素 ( ACTH)单克隆抗体 ,拟建立高灵敏度 ACTH- IRMA或 EL ISA。方法 ACTH1 - 1 3、ACTH1 - 2 8、ACTH 1 - 39和 ACTH1 8- 39分别与牛血清白蛋白偶联制备免疫原 ,在背部皮下和腹腔注射免疫 BAL B/ c鼠。按常规方法进行细胞融合 ,以 RIA法检测培养上清液 ,阳性孔经亚克隆 ,获得 2株分泌特异性抗体的杂交瘤细胞株 ;瘤细胞种于预致敏的BAL B/ c鼠腹腔 ,批量生产单抗性腹水 ;琼脂扩散法鉴定抗体类型 ;利用标准曲线的参数计算单抗的亲合常数 ;亲合纯化单抗并进行抗体固化试验。结果从 ACTH1 - 2 8免疫鼠获得单抗株为 5 G2 ,从 ACTH1 - 1 3免疫鼠获得单抗株为 D3 ;单抗腹水 5 0 %结合率时腹水最终稀释度分别为 5× 10 3和 2× 10 3;抗体类型均为 Ig G1/λ;5 G2和 D3亲合常数分别为 5 .1× 10 9L M- 1 和 4 .9× 10 7L M- 1 ;抗体包被实验显示 5 G2和 D3的固化抗体量分别为 1.4μg和 8.8μg时结合率达到饱和。结论 5 G2和 D3可以用于构建灵敏的 ACTH- IRMA或 EL ISA。Objective To produce monoclonal antibodies and establish a sensitive ACT H IRMA or ELISA MethodsACT H 1 13 ?ACT H 1 28 ?ACT H 1 39 and ACT H 18 39 were conjugated to bovine serum albumi n and BALB/c mice were immunized Splenocytes and SP 2/0 myeloma cel ls were fused by PEG The positive hybridomas were subcloned Then the screened hybri domas were inoculated into the abdominal cavity of BALB/c mice so as to produce ascitic fluids The subtypes of antibodies were identified by agarose diffusio n The affinity constants were calculated by Xu's formula from parameters of st andard curve. ResultsThe hybridomas 5G 2 and D3 were screened from the imm uned mice with ACT H 1 28 and ACT H 1 13 , and their final titre of ascitic fluids with 50% binding were 5×10 3 and 2 ×10 3 The subtypes of antibodies were IgG1/λ chain The affinity constants of antibodies 5G2 a nd D3 were 5 1×10 9 LM -1 and 4 9 ×10 7 LM -1 T he saturated binding capacities of immobilized antibodies 5G2 and D3 were 1 4 μg and 8 8 μg Conclusion5G2 and D3 can be used to establish a sensitive IRMA or ELISA

关 键 词：促肾上腺皮质激素 单克隆抗体 ACTH ELISA ACTH-IRMA 

分 类 号：R392-33[医药卫生—免疫学]