可溶性人细胞分化抗原28分子酶联免疫试剂盒的研制及应用  

Preparation and application of ELISA for detecting human soluble antigen CD28 in sera

作　　者：孙中文[1] 邱玉华[1] 马泓兵[1] 石云杰[1] 陶然[2] 孙玮丽[1] 程刚[1] 张学光[1] 

机构地区：[1]苏州大学医学生物技术研究所,215007 [2]苏州卫生职业技术学院检验医学系

出　　处：《中华检验医学杂志》2006年第6期518-521,共4页Chinese Journal of Laboratory Medicine

基　　金：江苏省自然科学基金资助项目(BK2004203);江苏省卫生厅医学科技发展基金资助项目(J200502)

摘　　要：目的研制灵敏、特异和稳定的可溶性人细胞分化抗原28（sCD28）酶联检测试剂盒，并探讨其临床应用意义。方法取识别不同抗原位点的单抗8G8和2D5分别作为包被单抗和检测单抗，建立双单抗夹心-sCD28酶联免疫检测法（sCD28-ELISA），并绘制标准工作曲线。同时，测定36名健康人、23例系统性红斑狼疮（SLE）、38例类风湿性关节炎（RA）和62例Grave病患者血清中sCD28含量。结果研制的sCD28试剂盒最低检测下限为0．3ng／ml，具有良好线性关系的检测范围是0．5～16．0ng／ml；8G8单抗包被测定板后置4℃冰箱保存180d，加入可溶性CD28-Ig重组蛋白的回收率在93％～109％之间。SLE、RA和Grave病患者血清中sCD28含量分别为0．9～7．3ng／ml、0．3～6．8ng／ml、0．2～3．5ng／ml，明显高于健康人（0～1．1ng／ml，P〈0．01）。结论自制的sCD28试剂盒，能准确测定样本中sCD28含量，可为sCD28分子基础研究和临床疾病辅助诊断、疗效评估及预后判断提供有价值的生物学参数，具有良好应用前景。Objective To establish a sensitive, specific, stable ELISA assay for detecting human soluble antigens CD28 and explore its clinical values. Methods The hybridoma cell lines （clone： 2D5, 8G8） secreting anti-CD28 antibody were successfully obtained through B lymphocyte hybridoma technique. The mAb 8G8 was used as coating antibody, and the mAb 2D5 as labeling antibody to prepare the standard work curve. Then concentrations of soluble antigen CD28 in serum samples for healthy donors, SLE and RA were analyzed. Results ELISA method for measuring soluble antigen CD28 was successfully established. The detecting limit was 0. 3 ng/ml and the linear range of standard work curve was 0. 5 - 16. 0 ng/ml. The sCD28 concentration in sera from healthy donors, SLE patients, rheumatoid arthritis patients and Grave disease patients were 0 - 1.1 ng/ml, 0. 9 - 7. 3 ng/ml,0. 3 - 6. 8 ng/ml,0. 2 - 3.5 ng/ml, respectively. Significant differences were revealed between healthy donors groups and patients groups （ P 〈 0. 01 ）. Conclusion Establishment of detecting sCD28 assay may be of significance in basic research and the diagnosis, curative effects evaluation and prognosis estimation of sCD28 related diseases.

关 键 词：可溶性人细胞分化抗原 酶联免疫吸附测定 

分 类 号：R446.6[医药卫生—诊断学]