HIV抗原抗体化学发光免疫检测方法的建立及优化

Establishment and Evaluation of a Two-Step Microplate Chemiluminescence Immunoassay to Detect HIV-1/2 Antibody and P24 Antigen

目的:建立并优化HIV抗原抗体化学发光免疫检测方法(CLIA)。方法:采用化学发光免疫的方法,通过对包被条件、HIV酶用量、反应加入量、反应时间和温度等反应参数进行优化,建立HIV抗原抗体联合检测方法,使用HIV国家参考品评价所建立方法的准确性。结果:包被比例取HIV-1抗原1∶5 000、HIV-2抗原1∶1 0000、HIV P24单抗1∶2 000;检测酶比例取Biotin-P24多抗1∶2 000;HRP-HIV-1抗原1∶5 000、HRP-HIV-2抗原1∶20 000、SA-HRP 1∶4 000;待测样本75μL/孔;第一步反应温度和时间分别为37℃和60 min,第二步反应温度和时间分别为37℃和30 min,为检测方法的较佳反应条件。检测国家参考品结果表明,阴性参考品符合率、阳性参考品符合率、灵敏度和精密性均符合国家参考品标准要求。结论:成功建立了特异、灵敏的HIV抗原抗体化学发光免疫检测方法,为在临床上推广应用提供了科学依据。

Objective:To establish and optimize the chemiluminescence immunoassay(CLIA)method for HIV antigen and antibody.Methods:A CLIA is used to establish a joint detection method for HIV antigen and antibody by optimizing the reaction parameters such as the coating conditions,the amount of HIV enzyme,the amount of reaction added,the reaction time and temperature,and the accuracy of the established method is evaluated using the HIV national reference.Results:The better reaction conditions:coating condition HIV-1 antigen 1∶5000,HIV-2 antigen 1∶10000,and HIV P24 mAb 1∶2000;Biotin-P241∶2000,HRP-HIV-1 antigen 1∶5000,HRP-HIV-2 antigen 1∶20000,SA-HRP 1∶4000;the first step reaction temperature and time were 37℃and 60min,and the second step was 37℃and 30min.The results show that the efficiency of the CLIA reagents met the requirement of the Chinese national reference standard.Conclusion:A specific chemiluminescence immunoassay method for HIV antigen and antibody has been successfully established,which provides a scientific basis for clinical application.