摘要
目的建立一种基于FopA单抗的阻断ELISA抗体检测方法。方法以pET-32a为载体表达土拉弗朗西斯菌的FopA蛋白,并以其免疫BALB/c小鼠制备单抗,以LVS超声裂解液为包被抗原、HRP标记FopA单抗为阻断抗体,建立直接阻断ELISA方法,利用阴性血清平均值+2SD或3SD确定该方法临界值,测定该方法特异性、敏感性、重复性及其与平板凝集试验的符合率,并利用该方法检测攻毒小鼠血清抗体变化趋势。结果筛选获得1株稳定分泌IgG1(κ型)单抗的杂交瘤细胞,并基于该单抗建立阻断ELISA方法,包被抗原最优浓度为3μg/mL、HRP标记FopA单抗最优稀释度为1∶4000、待检血清最优稀释度为1∶10,阴性临界值为27%、阳性临界值为41%、可疑区间为27%~41%,该方法的特异性、敏感性、重复性和符合率均良好,可检出攻毒9 d后小鼠血清抗体。结论成功制备FopA单抗,并基于该单抗建立一种快速、准确和高通量抗体检测的阻断ELISA方法,为我国土拉弗朗西斯菌流调和防控提供技术支撑。
This study was aimed at establishing a blocking ELISA method for antibody detection based on FopA monoclonal antibodies.FopA from Francisella tularensis was expressed with the pET-32a vector and used to immunize BALB/c mice to prepare monoclonal antibodies.A direct blocking ELISA was established by using LVS ultrasonic lysate as a coating antigen and HRP labeled FopA monoclonal antibodies as a blocking antibody.The specificity,sensitivity and replicability of this method were determined,and the coincidence rate with the plate agglutination test was assessed.A hybridoma cell line with stable secretion of IgG1(κ-type)monoclonal antibody was screened,and a blocking ELISA method based on this monoclonal antibody was established.The optimal concentration of coating antigen was 3μg/mL,the optimal dilution of HRP labeled FopA monoclonal antibody was 1∶4000,and the optimal dilution of tested serum was 1∶10.The negative cut off value was 27%,the positive cut off value was 41%,and the interval of 27%-41%indicated suspicion.The specificity,sensitivity,repeatability and coincidence rate of the method were all good.Serum antibodies in mice were detectable 9 days post challenge.The results indicated that the FopA monoclonal antibodies were successfully prepared,and used to establish a rapid,accurate,high-throughput blocking ELISA for antibody detection,thus laying a foundation for epidemiological surveying and control of Francisella tularensis in China.
作者
崔国林
戚心怡
吴村
蔡梦雷
张政钢
赵东旭
翟新国
CUI Guo-lin;QI Xin-yi;WU Cun;CAI Meng-lei;ZHANG Zheng-gang;ZHAO Dong-xu;ZHAI Xin-guo(College of Life Sciences and Food Engineering,Hebei University of Engineering,Handan 056038,China)
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2023年第9期850-856,共7页
Chinese Journal of Zoonoses
基金
河北省高等学校科学技术研究项目(No.QN2019015)
河北省自然科学基金项目(No.C2019402120)
国家重大基础研究项目(973项目)(No.2012CB518702)联合资助。
