基于磁微粒的丙型肝炎病毒抗体快速检测方法的建立

Establishment of magnetic particle-based rapid detection of hepatitis C virus antibodies

目的开发一种基于磁珠、吖啶酯以及自制生物素化丙型肝炎病毒(HCV)抗原的新型快速诊断方法。方法将生物素化HCV合成抗原包被于磁性微粒,加入待检样品后,再加入吖啶酯标记的兔抗人二抗,经洗涤后加入激发液,激发吖啶酯发出光粒子,并通过专有设备进行检测,评估方法的灵敏度、重现性和稳定性;采用国家血清盘评估方法的准确性,比较与时间分辨免疫荧光法检测试剂盒的一致性。结果建立的基于磁微粒的HCV检测方法,设备样本反应时间/吖啶酯标记反应时间设置为5min/5min;在灵敏度、阴阳性符合率等方面皆符合国家HCV血清盘的质量要求;试剂盒总的变异系数CV高、中、低质量浓度质控分别为4.0%、4.6%、7.6%;试剂盒在37℃存放7d,发光值及S/Co值的变动幅度皆<15%;与时间分辨免疫荧光法一致性Kappa值为0.949,具有较高的一致性。结论建立的基于磁微粒的HCV抗体快速检测方法快速、灵敏,具有良好的市场化应用前景。

Objective To develope a new rapid diagnostic method based on magnetic beads\\acridine ester\\biotinylated hepatitis C(HCV)antigen.Methods Biotinylated HCV synthetic antigen was coated on magnetic particles,the sample was added then rabbit anti-human secondary antibody labeled by acridine ester was added.After washing,stimulate liquid was added to induce acridine ester to emit light particles,which were detected by certain proprietary equipment.The sensitivity,reproducibility and stability of the method were evaluated;while the results were compared with those by time-resolved immunofluorescence method using national serum disk.Results For the established magnetic particle-based HCV detection method,the reaction time of equipment sample/acridine ester mark reaction time was set as 5 min/5 min.It met the quality requirements of national HCV serum disk in terms of sensitivity and compliance for both positive and negative rates.The general CV of the kit was 4.0%,4.6%and 7.6%for high,medium and low concentration QC,respectively.After 7 dstoreage at 37℃,the change range of luminescent value and S/CO value were less than 15%.Compared with time-resolved immunofluorescence,the kappa value was 0.949 for 2 methods,demonsrating high consistency.Conclution The established rapid detection method of HCV antibody based on magnetic particle is fast and sensitive,which has a good market application prospect.