检测土拉弗朗西斯菌的生物素—亲和素ELISA法的建立

    检测土拉弗朗西斯菌的生物素—亲和素ELISA法的建立

    景滢滢1,2 杨 宇2 王 静2 杨永莉2 孙肖红2 胡孔新2 王振东1

    （1.沈阳农业大学生物科学技术学院，沈阳 110866；2.中国检验检疫科学研究院，北京100025）

    摘要 〔目的〕 建立一种快速、敏感、特异检测土拉弗朗西斯菌的免疫学方法。〔方法〕应用生物素-亲和素标记系统，建立双抗体夹心ELISA方法，检测土拉弗朗西斯菌, 并进行“模拟样品”的检测判定其定量检测能力。〔结果〕建立的生物素—亲和素ELISA检测土拉弗朗西斯菌的方法具有很好的敏感性和特异性，检测FopA蛋白的灵敏度达到6ng/ml；批内变异系数1.96%～6.64%，批间变异系数8.94%～10.02%，重复性好；检测不同浓度马秋波GP2蛋白、天花M1R蛋白、黄热YF-E-D3蛋白、禽流感NP蛋白、登革病毒Deng-E-D3蛋白和普氏立克次体120N蛋白，未发现交叉反应；对“模拟样品”中土拉弗朗西斯菌FopA蛋白的检测回收率为91.7%～125.0%。〔结论〕应用生物化的FopA多抗与酶标亲合素结合，建立了检测土拉弗朗西斯菌的生物素—亲和素放大酶联免疫吸附法（BSA-ELISA），具有较好的灵敏度和特异性，在病原菌的识别、快速检测、应对生物恐怖威胁中具有广阔的应用前景。

    关键词土拉弗朗西斯菌；FopA蛋白；多克隆抗体；生物素—亲和素；放大酶联免疫吸附法

    〔中图分类号〕　R515.9 R446.61　〔文献标识码〕　B

    Establishment of a Biotin-avidin ELISA Methodfor Detection of Francisella Tularensis    JingYingying1, Yang Yu2, Wang Jing2，Yang Yongli2, Sun Xiaohong2, HuKongxin2, Wang Zhendong1. (1 College of Bioscience andBiotechnology, Shenyang Agricultural University, Shenyang 110866,China; 2 Chinese Academy of Inspection and Quarantine, Beijing100025, China)

    [Abstract]   Objective  To establish a rapid, sensitive and specific method for detectionof Francisella tularensis.   Methods  Biotin-avidin labeling system was adopted in the establishment of adouble antibody sandwich ELISA method for detection of Francisellatularensis. “Simulated samples” were tested to determine thequantitive detection capability.  Results    The new Biotin-avidin ELISA method, candetect 6 ng/ml of Francisella tularensi FopA protein with theintra-coefficient of variation1.96%~6.64%, and inter-coefficient ofvariation 8.94%~10.02%,, indicated the good sensitivity,specificity and, repeatability; the test results showed differentconcentrations of Machupo GP2 protein, Variola M1R protein, Yellowfever YF-E-D3 protein, avian influenza NP protein, Dengue feverDeng-E-D3 protein and Rickettsia Prowazeki 120N protein hand nocross reaction, analysis of “simulated sample” with Francisellatularensis FopA protein in food showed the recovery rate were91.7%~125.0%.  Conclusion   A biotin-avidinamplification enzyme-linked immunosorbent assay (BSA-ELISA) methodis established using biotechnology-oriented FopA polyclonalantibody and avidin-HRP for Francisella tularensis detection. thenew method will play an important part in the early identification,rapid detection of the pathogens and has broad applicationprospects in coping with the threaten of bioterrorism factor.

    [Key words]   Francisellatularensis; FopA protein; Polyclonal antibody; Biotin-avidin；Amplification enzyme-linked immunosorbent assay