摘要
目的 研制鼠疫菌F1抗体双抗原夹心ELISA诊断试剂盒,并进行验证。方法 用纯化的鼠疫菌F1抗原作为包被抗原,HRP标记的F1抗原作为酶标抗原,建立鼠疫菌F1抗体双抗原夹心ELISA检测方法,连续制备3批诊断试剂盒,并建立内部质控品。对试剂盒进行板内板间精密性、敏感性、特异性、准确性和稳定性验证。结果 鼠疫菌F1抗原的最佳包被浓度为0.6μg/ml,HRP标记的F1抗原的最佳工作浓度为1︰7 000。制备的试剂盒检测精密性质控品的板内变异系数为6.5%,板间变异系数为7.1%;检测高、中、低浓度内部质控品和精密性质控品的回收率在95%～112%之间;检测阳性血清和阴性血清的敏感性为100%;与正常人血清、正常兔血清、正常小鼠血清、兔抗假结核耶尔森菌血清、小鼠抗假结核耶尔森菌血清均无交叉反应;试剂盒稳定性良好,有效期至少为1年。结论 成功制备了鼠疫菌F1抗体双抗原夹心ELISA诊断试剂盒,达到了体外诊断试剂的注册要求,可用于鼠疫的临床监测、诊断及预后判断。
Objective To develop and verify a double antigen sandwich ELISA kit for Yersinia pestis F1 antibody. Methods The double antigen sandwich ELISA kit for Y. pestis F1 antibody was developed using purified F1 antigen as coating antigen and HRP-labeled F1 antigen as enzyme labeled antigen. Three batches of kits were prepared, and internal reference was set up. The kit was verified for precisions in intra- and inter-assays, sensitivity, specificity, accuracy and stability. Results The optimal concentration of F1 antigen for coating was 0. 6 p,g/ml, while the optimal working concentration of HRP-labeled F1 antigen was 1 ： 7 000. The CVs of results in intra- and inter-assays on reference for preci- sion were 6. 5% and 7. 1% respectively. The recovery rates of internal reference and the reference for precision at high, moderate and low concentrations were 95% - 112%. Both the sensitivities of the kit for positive and negative sera were 100%. No cross reactions with normal human serum, normal rabbit serum, normal mouse serum as well as rabbit and mouse sera against Y. pseudotuberculosis were observed. The kit showed high stability, of which the validity period was at least 12 months. Conclusion The double antigen sandwich ELISA kit for Y. pestis F1 antibody was successfully devel- oped, which met the requirements for license of in vitro diagnostic kit, and was suitable for the clinical monitoring, diagnosis and prognosis of plaque.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第12期1801-1804,共4页
Chinese Journal of Biologicals
基金
国家"863"课题(2006AA02Z461)
