毕赤酵母及大肠埃希菌表达的重组乙型肝炎病毒e抗原在乙型肝炎病毒e抗体检测中的比较分析  

Suitability of yeast and Escherichia coli expressed hepatitis B virus e antigen derivatives for detection of anti-HBe antibodies

作　　者：洪国强[1] 李朝霞[1] 陈月燕[1] 胡波[1] 李林[1] 

机构地区：[1]中山大学附属第三医院检验科,广州市510630

出　　处：《中华实验和临床感染病杂志（电子版）》2008年第4期255-263,共9页Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition)

基　　金：广东省科技计划项目(2006B36001010)

摘　　要：目的探讨毕赤酵母及大肠埃希菌表达的重组乙型肝炎病毒e抗原(recombinant hepa-titis B e antigen,rHBeAg)在乙型肝炎病毒e抗体(hepatitis B e antibody,HBeAb)免疫检测中的应用。方法用分子筛纯化两种rHBeAg,进行特异性和免疫反应性鉴定;分别用纯化后毕赤酵母表达的HBeAg(A)、大肠埃希菌表达的HBeAg(B)作为中和试剂,单克隆HBeAb为固相抗体,辣根过氧化物酶标记多克隆HBeAb为探测抗体,用固相-液相竞争抑制法酶联免疫吸附实验检测HBeAb标准品及乙型肝炎病毒PCR阳性和阴性的临床标本。结果(1)检测中国药品生物制品检定所HBeAb国家参考品结果:试剂A:阴性符合率(-/-)15/15,阳性符合率(+/+)10/10;灵敏度参考品最低检出量(稀释度)1#=1∶128,2#=1∶128,3#=1∶256。试剂B:阴性符合率(-/-)15/15;阳性符合率(+/+)9/10;灵敏度参考品最低检出量(稀释度)1#=1∶32;2#=1∶64;3#=1∶64;大肠埃希菌抗原的灵敏度明显低于毕赤酵母抗原;(2)对乙型肝炎病毒PCR阳性及阴性的临床标本进行检测:试剂A和试剂B对PCR阳性组的HBeAb阳性检出率分别为50.4%和51.6%,差异无显著统计学意义(P>0.05);PCR阴性组的HBeAb阴性检出率,试剂A和试剂B分别为91.2%和86.7%,差异有显著统计学意义(P<0.01)。结论毕赤酵母表达的HBeAg用于HBeAb的检测灵敏度及特异性均优于大肠埃希菌表达产物。Objective To explore the suitability of recombinant HBeAg (rHBeAg) expressed by Pichia pastoris and E.coli for immunoassay of anti-HBe antibody (HBeAb).Methods Yeast-derived HBeAg and E.coli-derived HBeAg were purified by gel filtration.The preparation of yeast-derived HBeAg (kit A) and E.coli-derived HBeAg (kit B) were served respectively as the neutralizing reagents,in combination with the monoclonal HBeAb as the solid-phase antigen and horseradish peroxidase(HRP)-labeled polyclonal HBeAb as the probe,to detect HBeAb standard panel of Chinese National Institute for the Control of Pharmaceutical and Biological Products and clinical samples including hepatitis B virus PCR positive and negative specimens selected by competitive inhibition ELISA.Results Detection of HBeAb standard by kit A showed that the negative rates (-/-)in negative standard and positive rates(+/+)in positive standard were 15/15 and 10/10,respectively.The dilution titres for 1#,2#,3# sensitivity standard were 1∶128,1∶128 and 1∶256,respectively;while detection by kit B showed that the negative rates in negative standard and the positive rates in positive standard were 15/15 and 9/10,respectively.The dilution titres for 1#,2#,3# sensitivity standard were 1∶32,1∶64 and 1∶64,respectively.The sensitivities of E.coli-derived HBeAg were obviously lower than P.pastoris-derived HBeAg in detection of HBeAb standard.HBeAb positive rate detection of clinical samples in PCR positive group by kit A and kit B were 50.4% and 51.6%,respectively.The difference was not significant between kit A and B (P>0.05);HBeAb negative rate in PCR negative group were 91.2% and 86.7% by kit A and kit B,and the difference was significant (P<0.01).Conclusions Compared with E.coli-derived HBeAg,P.pastoris-derived HBeAg optimized immunoassay HBeAb in sensitivity and specificity.

关 键 词：乙型肝炎病毒E抗原 基因表达 毕赤酵母 酶联免疫吸附实验 

分 类 号：R446.6[医药卫生—诊断学]