摘要
目的研究建立基于HIV免疫磁分离前处理技术的尿液HIV抗体高敏感性检测方法。方法采用羧基磁珠和HIV特异性抗原通过碳二亚胺缩合反应,优化制备HIV免疫磁珠;采用BCA法对磁珠偶联前后的HIV抗原浓度进行测定,采用ELISA法对HIV免疫磁珠捕获前后的样本中HIV抗体吸光度(OD)值进行检测,采用HRP标记抗原对免疫磁珠捕获后的HIV抗体进行OD值检测,以抗原偶联率与HIV免疫磁珠的抗体捕获效率为指标,建立HIV免疫磁珠在尿液样本中的前处理体系与检测方法;使用100份阴性尿液样本进行Cut-off值设定;使用3套血液确证HIV抗体阳性尿液2^(1)~2^(10)系列稀释样本进行方法敏感性验证;使用100份血液确证HIV抗体阳性尿液与100份健康人群尿液进行方法初步应用。结果300 nm羧基磁珠在100∶4的投料比下制备的HIV免疫磁珠的抗原偶联率为79.0%;10μg/mL HIV免疫磁珠在1 mL和5 mL尿液样本中室温捕获15~30 min后的抗体捕获效率为80.42%~93.99%;基于HIV免疫磁珠前处理技术的高敏感性检测方法Cut-off值为0.56;对3套2^(1)~2^(10)系列稀释HIV抗体尿液样本的检测敏感性较常规ELISA提高16~512倍;该方法对100份血液确证HIV抗体阳性的尿液检出数(81)高于常规ELISA检出数(52);全部验证与应用实验中均未发现阴性样本的假阳性现象。结论本研究建立的HIV免疫磁珠制备体系与HIV免疫磁珠尿液样本前处理技术,均具有良好的应用稳定性与检测有效性,基于HIV免疫磁分离技术的尿液HIV抗体检测方法敏感性高于目前ELISA检测方法。
Objective To establish a high sensitivity detection method for HIV antibody in the urine based on HIV immunomagnetic separation pretreatment technology.Methods We used the carboxyl magnetic beads and HIV-specific antigen to the preparation of HIV immunomagnetic beads by carbon diimine condensation reaction,the BCA method to determine the antigen concentration before and after magnetic bead coupling.The ELISA method was used to detect the OD value of HIV antibody in the samples before and after the capture of HIV immunomagnetic beads and the HRP labeled antigen was used to detect OD value of HIV antibody with immunomagnetic beads after capture.We also established the pretreatment system and detection method of HIV immunomagnetic beads in simulated urine samples,which was based on the antigen coupling rate and the antibody capture efficiency of HIV immunomagnetic beads.We used100 negative urine samples to set the cut-off value of the method and three sets of 21-210 diluted urine samples from confirmed blood ani-HIV positive infection to verify the sensitivity of the method.Finally,a total of 100 urine samples from confirmed blood aniHIV positive infection and 100 urine samples from healthy people were used for the method application.Results The antigenic coupling rate with the 300 nm carboxyl beads prepared at the input ratio of 100∶4 was 79.0%.The antibody capture efficiency of 10μg/m L HIV immunomagnetic beads in 1 m L and 5 m L urine samples was 80.42%-93.99%after 15-30 minutes capture at room temperature.The cut-off value of the high sensitivity detection method was 0.56,and the detection sensitivity of three sets of series diluted HIV antibody urine samples was increased 16-512 times compared to conventional ELISA.The detection number of clinically known HIV antibody positive urine samples(81)was higher than the conventional ELISA(52),and no false-positive phenomenon of 100 negative samples was found in all validation and application experiments.Conclusion We prepared the HIV immunomagnetic bead and established the HIV immunomagnetic bead urine sample pre-treatment techniques,which were both with good application stability and detection effectiveness.The sensitivity of urine HIV antibody detection method based on HIV immunomagnetic separation was higher than conventional ELISA detection methods.
作者
赫晓霞
李静雯
杜美红
康福英
陈尔凝
蒋岩
金聪
HE Xiaoxia;LI Jingwen;DU Meihong;KANG Fuying;CHEN Erning;JIANG Yan;JIN Cong(Beijing Engineering Research Center of Food Safety Analysis,Beijing Engineering Technology Research Centre of Gene Sequencing and Gene Function Analysis,Beijing Center for Physical&Chemical Analysis,Beijing 100089,China;National Center for AIDS/STD Control and Prevention,China CDC,National HIV/AIDS Reference Laboratory,NARL,Beijing 102206)
出处
《中国艾滋病性病》
CAS
CSCD
北大核心
2021年第10期1059-1064,共6页
Chinese Journal of Aids & STD
基金
北京市理化分析测试中心改革与发展专项课题(2021ZL0110)
北京市科学技术研究院改革发展培育项目(PY2020JK43)
国家自然科学基金(31972146)。
关键词
免疫磁分离
艾滋病病毒抗体
尿液
高敏感检测
Immunomagnetic separation
HIV antibody
Urine
Highly sensitive detection
