检测可溶型CD226双mAb夹心ELISA的建立及初步应用  

Development and preliminary application of a double mAb sandwich ELISA for detecting soluble CD226

作　　者：宁双飞[1] 贾卫[1] 张新海[1] 杨琨[1] 李琦[1] 刘雪松[1] 金伯泉[1] 

机构地区：[1]第四军医大学免疫学教研室,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2003年第2期192-194,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家重点基础研究发展规划资助项目 (No.2 0 0 1CB51 0 0 0 4 );国家自然科学基金重点资助项目 (No.30 0 30 1 30 )

摘　　要：目的 :建立一种检测可溶型CD2 2 6 (sCD2 2 6 )的双mAb夹心ELISA ,并用于临床标本的检测。方法 :确定包被mAbLeoA1和HRP mAbFMU3的最佳工作浓度及最佳稀释液后 ,建立双mAb夹心ELISA ,并对方法的特异性、敏感性和稳定性进行鉴定。再以建立的方法检测临床标本。结果 :包被mAbLeoA1的最佳工作浓度为 2 .5mg/L ,HRP mAbFMU3的最佳工作浓度 1∶4 0 0 ,标准品及HRP mAb的最佳稀释液分别为 1g/LBSA 1mL/LTween2 0 PBS和 30 g/LPEG PBS .建立的双mAb夹心ELISA ,其特异性 (与人IgG无交叉反应 ,阻断实验中阻断效应呈剂量依赖性 )、敏感性 (检出下限为 110ng/L标准品CD2 2 6 /Fc融合蛋白 )及稳定性 (CV <10 .2 % )均较良好。对部分临床标本的检测中 ,发现 6例类风湿关节炎(RA)患者血清sCD2 2 6的水平升高 ,与正常人血清sCD2 2 6水平相比差异显著 (P <0 .0 1)。结论 :建立一种特异性高、重复性好、较敏感的检测sCD2 2 6的双mAb夹心ELISA 。AIM: To develop a double mAb sandwich ELISA for detecting sCD226 and apply it to clinical specimens. METHODS: The optimal concentrations of coating mAb and HRP mAb, and the optimal diluents were determined. A double mAb sandwich ELISA then was established. The specificity, sentivity and stability of the method were identified. And clinical specimens were detected by this method. RESULTS: The optimal concentrations of coating mAb LeoA1 and HRP mAb FMU3 were 2.5 mg/L and 1∶400, respectively, and the optimal diluents for standard antigen and HRP mAb were 1 g/L BSA 1 mL/L Tween20 PBS and 30 g/L PEG PBS.The ELISA achieved satisfactory results in respect to specificity (no cross reaction to human IgG, in block test blocking effect was does dependent), sensitivity (the detected threshold was 110 ng/L) and stability (CV<10.2%). Statistical analysis showed that the serum sCD226 level in patients with rheumatoid arthritis (RA) was significantly higher than that in healthy adults. CONCLUSION: A double mAb sandwich ELISA with high specifity, and relative sensitivity for detection of sCD226 has been developed. It is shown to be an applicable method of detecting sCD226 in clinical specimens.

关 键 词：可溶型CD226 单克隆抗体 酶联免疫吸附试验 临床应用 类风湿性关节炎 

分 类 号：R446.61[医药卫生—诊断学] R593.22[医药卫生—临床医学]