CAJ | 학술논문

目的研究用戊型肝炎病毒4型(HEV4-)开放读码框架(ORF)2主要免疫表位截短多肽排除抗-HEV IgM检测中发生假阳性的可行性.方法用重组HEV-4 ORF2蛋白主要抗原决定簇多肽(459～607位氨基酸)及其截短多肽(472～607位氨基酸)为抗原分别包被ELISA板,用间接ELISA法分别检测35份HEV RNA阳性患者血清、69份健康人血清和117份可疑阳性血清标本的抗-HEV ISM,并用免疫印迹法(Western blot,WB)确认,逆转录(RT)-PCR检测HEV RNA加以比较.结果 WB检测结果显示,戊型病毒性肝炎患者血清中抗-HEV ISM仅与459～607多肽二聚体反应,而不与其单体及472～607多肽反应.间接ELISA法检测35份HEV RNA阳性患者血清抗-HEV IgM均能与459～607多肽反应,而不与472～607多肽反应;69名健康人血清与2种多肽都不反应.117份可疑阳性血清中,5份能同时与2种多肽反应,但450 nm吸光度(A450)值之差小于0.5.WB检测这5份血清抗-HEV IgM均阴性;RT-PCR检测HEV RNA为阴性,说明这5份血清为非特异性吸附引起的假阳性.结论 ORF2 459～607多肽可有效检测抗-HEV ISM,根据血清与2种多肽反应的A450差异,能有效排除因非特异性吸附导致的抗-HEV IgM假阳性.
목적 연구용무형간염병독4형(HEV4-)개방독마광가(ORF)2주요면역표위절단다태배제항-HEV IgM검측중발생가양성적가행성.방법 용중조HEV-4 ORF2단백주요항원결정족다태(459～607위안기산)급기절단다태(472～607위안기산)위항원분별포피ELISA판,용간접ELISA법분별검측35빈HEV RNA양성환자혈청、69빈건강인혈청화117빈가의양성혈청표본적항-HEV ISM,병용면역인적법(Western blot,WB)학인,역전록(RT)-PCR검측HEV RNA가이비교.결과 WB검측결과현시,무형병독성간염환자혈청중항-HEV ISM부여459～607다태이취체반응,이불여기단체급472～607다태반응.간접ELISA법검측35빈HEV RNA양성환자혈청항-HEV IgM균능여459～607다태반응,이불여472～607다태반응;69명건강인혈청여2충다태도불반응.117빈가의양성혈청중,5빈능동시여2충다태반응,단450 nm흡광도(A450)치지차소우0.5.WB검측저5빈혈청항-HEV IgM균음성;RT-PCR검측HEV RNA위음성,설명저5빈혈청위비특이성흡부인기적가양성.결론 ORF2 459～607다태가유효검측항-HEV ISM,근거혈청여2충다태반응적A450차이,능유효배제인비특이성흡부도치적항-HEV IgM가양성.
Objective To exclude false positivities in detection of IgM antibodies against hepatitis E vires of genotype 4 (HEV-4) using a truncated immunodominant polypeptide of HEV open reading frames (ORF2). Methods The recombinant ORF2 immunodominant polypeptide corresponding to amino acids (AA) 459-607 and a truncated polypeptide corresponding to AA 472-607 were separately applied to coat ELISA plates. Anti-HEV IgM from 35 serum samples with HEV RNA positive, 69 serum samples from healthy individuals and 117 clinically suspicious HEV RNA positive serum samples was detected by an indirect ELISA and was confirmed by western blot in protein level and RT-PCR detecting in RNA level. Results Western blot analysis showed that the sera from HEV patients reacted with the dimmer of peptide 459-607, but they didn't react with the monomer and peptide 472-607. The ELISA showed that all 35 serum HEV RNA positive samples reacted with peptide 459-607 but not with peptide 472-607 and none of the 69 serum samples from healthy individuals reacted with either polypeptide. Among 117 chnically suspicious HEV RNA serum samples, 5 samples reacted simultaneously with both polypeptides. But the difference between 450 nm absorbance (A450) value was less than 0. 5. Western blot analysis demonstrated that all the 5 serum samples were anti-HEV IgM- negative. The 5 serum samples was detected negative by RT-PCR, indicating that the false pesitivities were caused by non-specific absorption. Conclusions ORF2 peptide 459-607 may be used to detect anti-HEV lgM efficiently. The false positivities caused by non-specific absorption can be largely excluded according to the difference between 45Ohm absorbance (A450) value when serum reacts with both polypeptides.