人α1微球蛋白ELISA检测试剂盒的制备及初步应用  

Preparation and preliminary application of ELISA kit for human α1-microglobulin

作　　者：魏雁虹[1] 何巍[2] 耿辉[1] 宋帅[1] 刁斌斌[1] 杨广民[1] 

机构地区：[1]吉林省人民医院检验科,吉林长春130021 [2]长春生物制品研究所有限责任公司,吉林长春130062

出　　处：《中国生物制品学杂志》2014年第12期1560-1564,共5页Chinese Journal of Biologicals

基　　金：吉林省科技厅科技发展计划项目(20130102085jc);吉林省卫生厅自然科学基金(2012Z078)

摘　　要：目的制备人α1微球蛋白（alpha 1-microglobulin,α1-MG）ELISA定量检测试剂盒,并初步应用于临床标本的检测。方法用重组人α1-MG蛋白免疫小鼠,采用杂交瘤技术制备特异性单克隆抗体,对单抗的特性进行鉴定后,采用双抗体夹心ELISA方法制备α1-MG试剂盒,并进行线性范围、灵敏度、准确性、精密性验证。应用制备的试剂盒检测100份健康人血清中的α1-MG含量,计算正常参考值;分别应用试剂盒及放射性免疫分析法（radioactive immunoassay,RIA）检测87份人血清标本,分析试剂盒与RIA法检测结果的相关性。结果经间接ELISA法筛选及克隆化培养,共获得4株稳定分泌抗α1-MG单抗的杂交瘤细胞株;制备的试剂盒最佳线性范围为0.5-60 mg/L,灵敏度为0.03 mg/L,检测不同浓度α1-MG的回收率在92.7%-97.1%之间,批内、批间变异系数（CV）分别为4.78%-8.57%和4.30%-6.84%;该试剂盒检测健康人血清样本中α1-MG含量正常参考值为15-32 mg/L,与RIA法的检测结果具有良好的相关性（r=0.958 6）。结论制备的人α1-MG ELISA检测试剂盒具有较高的准确性和灵敏度,且与RIA法具有良好的相关性,符合临床免疫分析的要求,适用于临床检测和科研应用。Objective To prepare an ELISA kit for quantitative detection of alpha-1 microglobulin（α1-MG）and preliminarily apply to the test for clinical samples. Methods Mice were immunized with recombinant α1-MG to prepare monoclonal antibody（Mc Ab）by hybridoma technique. The characters of prepared Mc Ab were identified. The ELSIA kit for α1-MG was developed by double antibody sandwich ELISA technique,and verified for linear range,sensitivity,accuracy and precision. The α1-MG contents in 100 healthy human serum samples were determined by the prepared kit,of which the normal reference value was calculated. A total of 87 human serum samples were determined by the kit and radioimmunoassay（RIA）separately,and the relationship between the results was analyzed. Results Four hybridoma cell strains secreting Mc Abs against α1-MG were screened by indirect ELISA. The optimal linear range of prepared kit was0. 5 - 60 mg / L,while the sensitivity was 0. 03 mg / L,the recovery rates of α1-MG at various concentrations were92. 7% - 97. 1%,and the coefficients of variation（CVs）in intra- and inter-assays were 4. 78% - 8. 57% and 4. 30% -6. 84% respectively. The normal reference value of α1-MG content in healthy human serum samples determined by the prepared kit was 15 - 32 mg / L,which showed good relationship to that by RIA（r = 0. 958 6）. Conclusion The prepared ELISA kit for α1-MG showed high accuracy and sensitivity,by which the determination result showed good relationship to that by RIA. The kit met the requirements for clinical immune analysis and was suitable for clinical detection and scientific research.

关 键 词：Α1微球蛋白 酶联免疫吸附测定 试剂盒 参考值 

分 类 号：R692[医药卫生—泌尿科学] R446.61[医药卫生—外科学]