乙型肝炎病毒大蛋白时间分辨荧光免疫分析法的建立及应用

目的 建立定量检测患者血清HBV-大蛋白（LP）的TRFIA.方法 以抗人HBV-LP单克隆抗体和抗HBs多克隆抗体分别作为固相抗体和铕标记抗体,应用TRFIA和ELISA对标准品、66份慢性乙型肝炎患者血清、30份健康体格检查者血清中HBV-LP进行检测.分析TRFIA和ELISA的有效检测范围等,统计学比较采用χ^2检验和直线相关分析.结果 标准品TRFIA检测结果示该法剂量-反应曲线呈良好线性相关（r=0.999）.ELISA法测得的正常（30份健康者血清）95％参考范围为0～1.36 mg/L.TRFIA灵敏度（最小测定值）为0.10 mg/L,30份健康血清HBV-LP的检测结果均正常,特异性100％.66份患者血清TRFIA与ELISA检测结果的r为0.800 9（P＜0.001）,两者检测的阳性率差异有统计学意义[89.4％ （59/66）与77.3％（51/66）,χ^2=6.13,P＜0.01].TRFIA有效可测范围（CV＜ 10％）为1.35～2 764.00 mg/L,ELISA为10.80～691.00 mg/L.TRFIA回收率范围为95.93％ ～ 107.62％.结论 TRFIA法检测患者血清中的HBV-LP灵敏度和特异性高,检测范围宽,对早期筛选HBV患者及监测抗病毒治疗的疗效具有潜在的价值.

The establishment and application of a time-resolved fluoroimmunoassay in detection of HBV large surface protein

Objective To establish a method of TRFIA with high sensitivity and broad detecting range for serum HBV large surface protein （HBV-LP）.Methods The monoclonal antibody of HBV-LP was covered on the microwell plate and incubated with HBV-LP in blood sample,then Eu3＋ labeled antibody of HBs was added.HBV-LP in standard substance,blood samples of 66 chronic hepatitis B patients and 30 healthy controls was detected by the TRFIA and ELISA.χ^2 test and linear correlation analysis were used for data analysis.Results The dose-response curve of standard substance with TRFIA had good linear correlation （r=0.999）.Normal reference range was established at 0-1.36 mg/L based on the ELISA results of 30 healthy controls.The sensitivity was 0.10 mg/L.The specificity was 100％ （30/30）.Correlation coefficient between the TRFIA and the ELISA was 0.800 9 （P＜0.001）.The positive detecting rates of the 2 methods were significantly different （89.4％（59/66） vs 77.3％（51/66）,χ^2 =6.13,P＜0.01）.The recovery rate for HBV-LP was between 95.93％-107.62％.The effective detecting range（CV＜10％） of TRFIA was 1.35-2 764.00 mg/L,and that of ELISA was 10.8-691.0 mg/L.Conclusion The TRFIA was established for HBV-LP detection with higher sensitivity and wider detecting range compared to ELISA.It has potential value for HBV screening and monitoring of antiviral therapy.