González-Peralta R P, Fang J W, Davis G L, Gish R, Tsukiyama-Kohara K, Kohara M, Mondelli M U, Lesniewski R, Phillips M I, Mizokami M
Department of Medicine, University of Florida, Gainesville 32610.
J Hepatol. 1994 Jan;20(1):143-7. doi: 10.1016/s0168-8278(05)80481-7.
To optimize the detection of hepatitis C viral antigens in liver tissue, cryostat and formalin-fixed, paraffin-embedded liver sections from 21 patients with chronic hepatic C viral infection were studied. For cryostat sections, six different fixatives were compared. Sixteen primary antibodies were tested: nine different mouse monoclonal anti-hepatitis C virus-core antibodies, a human monoclonal anti-hepatitis C virus-non-structural 4, and six rabbit polyclonals directed against synthetic peptides of the hepatitis C virus core, envelope, and non-structural 3, non-structural 4, non-structural 5. Three detection systems, 3- and 5-step peroxidase-antiperoxidase and avidin-biotin complex, were examined. In cryostat sections, acetone/chloroform formation consistently produced the best signal-to-background ratio. Five anti-hepatitis C virus-core monoclonals which recognize amino acid sequence 26-45 of the hepatitis C virus-core region consistently detected the viral antigen, but not the monoclonals directed against 39-74 of the hepatitis C virus-core region. The human anti-hepatitis C virus-non-structural 4, which reacts to amino acid sequence 1700-1705, also regularly detected viral antigen. The rabbit polyclonals produced either negative or nonspecific staining. The 5-step peroxidase-antiperoxidase provided the strongest signal and the avidin-biotin system produced high background consistently. Overall, hepatitis C virus core and non-structural 4 antigens were detected in 71% and 57% of the patients studied. Of the 16 patients seropositive for hepatitis C virus RNA, 75% and 69% had detectable hepatitis C virus core and non-structural 4, in contrast to 60% and 20% of the five hepatitis C virus RNA seronegative patients.(ABSTRACT TRUNCATED AT 250 WORDS)
为优化肝组织中丙型肝炎病毒抗原的检测，对21例慢性丙型肝炎病毒感染患者的低温恒温器切片以及经福尔马林固定、石蜡包埋的肝脏切片进行了研究。对于低温恒温器切片，比较了六种不同的固定剂。测试了16种一抗：九种不同的小鼠单克隆抗丙型肝炎病毒核心抗体、一种人单克隆抗丙型肝炎病毒非结构蛋白4，以及六种针对丙型肝炎病毒核心、包膜、非结构蛋白3、非结构蛋白4、非结构蛋白5合成肽的兔多克隆抗体。检查了三种检测系统，即三步和五步过氧化物酶抗过氧化物酶以及抗生物素蛋白-生物素复合物。在低温恒温器切片中，丙酮/氯仿固定始终产生最佳的信噪比。五种识别丙型肝炎病毒核心区域氨基酸序列26-45的抗丙型肝炎病毒核心单克隆抗体始终能检测到病毒抗原，但针对丙型肝炎病毒核心区域39-74的单克隆抗体则不能。与人单克隆抗丙型肝炎病毒非结构蛋白4（对氨基酸序列1700-1705有反应）也能定期检测到病毒抗原。兔多克隆抗体产生阴性或非特异性染色。五步过氧化物酶抗过氧化物酶提供最强信号，抗生物素蛋白-生物素系统始终产生高背景。总体而言，在所研究的患者中，71%和57%检测到丙型肝炎病毒核心抗原和非结构蛋白4抗原。在16例丙型肝炎病毒RNA血清学阳性患者中，75%和69%可检测到丙型肝炎病毒核心抗原和非结构蛋白4，相比之下，五例丙型肝炎病毒RNA血清学阴性患者中这一比例分别为60%和20%。（摘要截短于250字）
