基孔肯雅病毒免疫球蛋白G抗体酶联免疫吸附实验快检试剂盒的研制与应用  

Development and application of a rapid IgG antibody ELISA kit for Chikungunya virus

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作  者:徐晓立 胡潇予 李春缘 曹孟涛 刘纪茹 刘炅 任瑞文 XU Xiaoli;HU Xiaoyu;LI Chunyuan;CAO Mengtao;LIU Jiru;LIU Jiong;REN Ruiwen(Instrumental Analysis&Research Center of South China Agricultural University,Guangzhou,Guangdong 510642,China;Center for Disease Control and Prevention of Southern Theater Command,Guangzhou,Guangdong 510507,China;Guangdong Arbovirus Diseases Emergency Technology Research Center,Guangzhou,Guangdong 510507,China)

机构地区:[1]华南农业大学测试中心,广东广州510642 [2]南部战区疾病预防控制中心,广东广州510507 [3]广东省虫媒病毒性传染疾病应急技术研究中心,广东广州510507

出  处:《中国热带医学》2024年第4期438-442,共5页China Tropical Medicine

基  金:全军实验动物专项[No.SYDW(2018)04];广东省科技计划项目(No.2016A020219006)。

摘  要:目的研制检测基孔肯雅热免疫球蛋白G(immunoglobulin G,IgG)抗体的酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)快检试剂盒,为流行病学调查及实验室诊断提供新的工具。方法以基孔肯雅病毒特异性原核表达抗原pMal-chik23为诊断抗原,以辣根过氧化物酶(horseradish peroxidase,HRP)标记抗IgG二抗为显色抗体,正交实验对诊断抗原、待检血清、二抗工作浓度进行优化,并对包被、封闭、孵育及显色等反应条件进行系统优化,在临床大样品评估的基础上,确定ELISA检测的临界值。在此基础上,对ELISA反应的特异性、灵敏性及稳定性进行评估,研制组装检测基孔肯雅热IgG抗体的ELISA快检试剂盒。结果经系统优化,研制、组装了检测基孔肯雅热IgG抗体的间接ELISA快检试剂盒,其最佳反应条件为:1.0μg/mL包被抗原经碳酸盐缓冲液4℃包被24 h,HBV封闭液37℃封闭4 h,待检样品1∶101稀释后,100μL/孔,37℃反应40 min,磷酸盐吐温缓冲液(phosphate buffered saline with tween 20,PBST)洗涤4次,HRP标记兔抗人IgG 1∶20000、HRP标记羊抗鼠IgG 1∶10000稀释后,100μL/孔,37℃反应30 min,PBST洗涤5次,TMB显色液100μL,37℃显色10 min,50μL 20%H2SO4终止反应,双波长450/630 nm测定A450 nm值。试剂盒性能评估结果表明,所研制试剂盒与所试阳性样品检测A450 nm值>0.43,阴性样品检测A450 nm值<0.04,S/N比值>10,与辛德毕斯、盖塔、罗斯河、登革等9种其他相近虫媒病毒均无交叉反应,并具较高稳定性,板内变异系数为0.76%~2.12%,板间变异系数为0.64%~1.85%,批间变异系数为0.83%~2.31%,均小于3%,4℃保存1年性能无明显下降。结论研制了检测基孔肯雅热IgG抗体的间接ELISA快检试剂盒,具备良好的灵敏性、特异性与稳定性。Objective To develop an ELISA kit to detect IgG antibodies of Chikungunya virus(CHIKV),providing a new method for epidemiological investigation and detection in the field for CHIKV infection.Methods Using the CHIKV-specific recombinant protein pMal-chik23 as diagnostic antigen,HRP-labeled anti-IgG antibody as color-developing antibody,and the working concentration of diagnostic antigen,serum to be tested and second antibody were optimized using orthogonal.The reaction conditions of ELISA reaction,such as coating,blocking,incubation,and color-developing were systematically optimized.The cut-off value for ELISA detection was established based on the assessment of a large clinical sample set.On this basis,the specificity,sensitivity,and stability of the ELISA response were evaluated to develop and assemble a rapid ELISA kit for the detection of Chikungunya fever IgG antibodies.Results On the basis of systematic conditions optimization,an indirect ELISA kit for the detection of IgG antibodies against CHIKV was developed and assembled.The optimal reaction conditions were identified as 1.0μg/mL antigen was coated using carbonate buffer at 4℃for 24 hours.Then the microplate was blocked using HBV blocking solution at 37℃for 4 hours.100μL/well samples to be tested were diluted at 1∶101,reacted at 37℃for 40 minutes,and washed 4 times with PBST.Thus,HRP-labeled rabbit anti-human IgG was diluted at 1∶20000,HRP-labeled sheep anti-mouse IgG was diluted at 1∶10000,reaction at 37℃for 30 minutes,and washed 5 times with PBST.Finally,100μL/well TMB solution was added and incubated at 37℃for 10 minutes.Then terminate the reaction with 50μL of 20%H2SO4 and measure the A450 value at dual wavelengths of 450/630 nm(A450).The evaluation results showed that ELISA A450 of Chikungunya fever-positive samples were more than 0.43,while the ELISA A450 of negative samples was less than 0.04,and the S/N ratio>10.Specificity test showed that the developed kit had no cross-reaction with 9 other similar arbovirus species such as

关 键 词:基孔肯雅热 快速检测 酶联免疫吸附实验 

分 类 号:R373[医药卫生—病原生物学]

 

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