胞内分枝杆菌ELISA检测方法的建立及初步应用
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摘要
目的建立胞内分枝杆菌双抗夹心ELISA检测胞内分枝杆菌生物学新方法。方法以自主制备的胞内分枝杆菌HBHA单克隆抗体为包被抗体,建立胞内分枝杆菌ELISA检测方法。应用棋盘法优化检测条件,同时试验对封闭液进行了最佳选择,对检测样品的特异性、敏感性和准确度进行评估。结果确定抗体包被最佳浓度为4μg/ml,抗鼠血清最佳稀释浓度为1:400,同时选择P/N值为3.38的1%BSA作为最佳封闭剂。双抗夹心法对新金分枝杆菌、耻垢分枝杆菌、浅黄分枝杆菌和卡介苗等抗原检测的P/N值均小于2.1,无交叉反应。当菌体浓度为200个/ml时,检测结果仍为阳性,应用该方法检测60份样品中,呈现阳性的菌株为56份,检测准确度高达93.3%。结论本方法可作为胞内分枝杆菌快速检测和流行病学调查的工具,同时对畜牧业的健康发展提供一种新的技术保障。
Objective To establish a new method for the detection of Mycobacterium intracellular by double-antibody sandwich ELISA.Methods An ELISA assay method was established by using an independently prepared Mycobacterium intracellular HBHA monoclonal antibody as a coating antibody.The checkerboard method was used to optimize the detection conditions,and the optimal selection of the blocking solution was carried out at the same time,and the specificity,sensitivity and accuracy of the test samples were evaluated.Results The optimal concentration of antibody coating was determined to be 4 μg/ml,and the optimal dilution concentration of anti-rat serum was 1∶400.At the same time,1% BSA with P/N value of 3.38 was selected as the optimal blocking agent.The P/N values of the double-anti-sandwich method for the detection of antigens such as Mycobacterium aureum,Mycobacterium smegmatis,Mycobacterium gilvum and BCG were less than 2.1,and there was no cross-reactivity.When the concentration of the cells was 200/ml,the test result was still positive.Of the 60 samples tested,56 were positive,and the detection accuracy was as high as 93.3%.Conclusion The method can be used as a tool for rapid detection and epidemiological investigation of intracellular mycobacteria,and provide a new technical guarantee for the healthy development of animal husbandry.
Objective To establish a new method for the detection of Mycobacterium intracellular by double-antibody sandwich ELISA.Methods An ELISA assay method was established by using an independently prepared Mycobacterium intracellular HBHA monoclonal antibody as a coating antibody.The checkerboard method was used to optimize the detection conditions,and the optimal selection of the blocking solution was carried out at the same time,and the specificity,sensitivity and accuracy of the test samples were evaluated.Results The optimal concentration of antibody coating was determined to be 4 μg/ml,and the optimal dilution concentration of anti-rat serum was 1∶400.At the same time,1% BSA with P/N value of 3.38 was selected as the optimal blocking agent.The P/N values of the double-anti-sandwich method for the detection of antigens such as Mycobacterium aureum,Mycobacterium smegmatis,Mycobacterium gilvum and BCG were less than 2.1,and there was no cross-reactivity.When the concentration of the cells was 200/ml,the test result was still positive.Of the 60 samples tested,56 were positive,and the detection accuracy was as high as 93.3%.Conclusion The method can be used as a tool for rapid detection and epidemiological investigation of intracellular mycobacteria,and provide a new technical guarantee for the healthy development of animal husbandry.
引文
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[10] 毛冉,张宇,王铁峰,等.牛源非结核分枝杆菌的分离培养与结果分析[J].河北科技师范学院学报,2010,24(4):32-35.
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[2] Rom WN,Garay SM.Tuberculosis[M].2nd ed.Philadelphia:Lippincott Williams & Wilkins,2003:651-662.
[3] Yildirim Z,Hacievliyagil S,Kutlu NO,et al.Effect of water extract of Turkish propolis on tuberculosis infection in guinea-pigs[J].Pharmacological Research,2004,49(3):287-292.
[4] 葛淑敏,张淑华,何秀霞,等.胞内分枝杆菌HBHA基因的克隆、分析及原核表达[J].西北农林科技大学学报:自然科学版,2014,42(4):7-14.
[5] 温晓艳,周芳,张彦霞,等.抗流感嗜血杆菌外膜蛋白P6单克隆抗体的制备及鉴定[J].细胞与分子免疫学杂志,2014,30(10):1051-1053,1061.
[6] 宋百军,刘原源,宫鹏涛,等.双抗夹心ELISA检测犬粪贾第虫抗原方法的建立及应用[J].中国病原生物学杂志,2014,9(10):902-904,907.
[7] 吴芬.田鼠巴贝虫FTA-LAMP及基于BmSA1和SRA5抗原的诊断技术建立[D].中国疾病预防控制中心,2016.
[8] 宋慧琦,杨升,高珺珊,等.检测犬弓形虫SAG3抗体间接ELISA方法的建立[J].中国病原生物学杂志,2016,11(4):338-341,345.
[9] 王燕,元振杰,曹旭东,等.结核分枝杆菌蛋白TB9.7间接ELISA方法的建立[J].家畜生态学报,2011,32(3):65-70.
[10] 毛冉,张宇,王铁峰,等.牛源非结核分枝杆菌的分离培养与结果分析[J].河北科技师范学院学报,2010,24(4):32-35.
[11] Koh WJ,Kwon OJ,Lee KS.Diagnosis and treatment of nontuberculous mycobacterial pulmonary diseases:A Korean perspective[J].Journal of Korean Medical Science,2005,20(6):913-925.
[12] 王铁峰,王照,李新殿.屠宰工作环境中非结核分枝杆菌流行情况[J].吉林农业,2016(1):82-83.
[13] 葛淑敏,王诚,王准,等.胞内分枝杆菌HSP65基因克隆、分析及其原核表达的研究[J].现代预防医学,2015,42(3):515-518,532.