Attallah Abdelfattah M, Ismail Hisham, Tabll Ashraf A, Shiha Gamal E, El-Dosoky Ibrahim
R&D Department, Biotechnology Research Center, New Damietta City, Egypt.
J Immunoassay Immunochem. 2003;24(4):395-407. doi: 10.1081/IAS-120025777.
The limitations of dominant methods-based on the detection of anti-HCV antibodies or HCV viremia currently used for the diagnosis of HCV infection enhance efforts to have a rapid, simple, sensitive, and specific alternative diagnostic approach to detect viral antigens. A highly reactive IgG antibody was raised to HCV-NS4 recombinant antigen. The produced antibody showed no cross-reactivity with the other HCV structural and nonstructural recombinant antigens (C1 + 2, C3 + 4, E2/NS1, NS3, NS5). The well established ELISA technique was adapted to detect the new target HCV-NS4 antigen in serum samples. Extremely high agreement was found between the results of ELISA and qualitative detection of HCV-RNA, using a RT-PCR test as a gold standard for the diagnosis of HCV infection. Based on these encouraging results, a novel enzyme immunoassay; dot-ELISA was developed for rapid (approximately 5 min) and simple qualitative detection of the target HCV antigen in serum. The developed method detected the HCV target antigen in 95% of serum samples from HCV infected individuals, with a specificity of 97% using sera of noninfected individuals in comparison with PCR test. The antigen detection method showed high predictive values of positive (99%) and negative (90%). Moreover, the dot-ELISA could detect the HCV target antigen in sera negative for anti-HCV Abs, but positive for HCV-RNA, and in sera of HCV infected individuals with low viremia, as well as those with high viremia, using quantitative RT-PCR. Accordingly, the developed highly sensitive and specific HCV antigen detection method could be applied for mass screening of HCV infection.
基于目前用于丙型肝炎病毒（HCV）感染诊断的抗HCV抗体或HCV病毒血症检测的主流方法存在局限性，这促使人们努力寻求一种快速、简单、灵敏且特异的替代诊断方法来检测病毒抗原。针对HCV-NS4重组抗原产生了一种高反应性的IgG抗体。所产生的抗体与其他HCV结构和非结构重组抗原（C1 + 2、C3 + 4、E2/NS1、NS3、NS5）无交叉反应。采用成熟的ELISA技术来检测血清样本中的新型靶标HCV-NS4抗原。以逆转录聚合酶链反应（RT-PCR）检测作为HCV感染诊断的金标准，发现ELISA结果与HCV-RNA定性检测结果之间具有极高的一致性。基于这些令人鼓舞的结果，开发了一种新型酶免疫测定法；斑点ELISA，用于快速（约5分钟）且简单地定性检测血清中的靶标HCV抗原。所开发的方法在95%的HCV感染个体血清样本中检测到了HCV靶标抗原，与PCR检测相比，使用未感染个体的血清时特异性为97%。该抗原检测方法显示出高的阳性预测值（99%）和阴性预测值（90%）。此外，斑点ELISA能够在抗HCV抗体阴性但HCV-RNA阳性的血清中，以及在病毒血症水平低和高的HCV感染个体血清中检测到HCV靶标抗原，采用定量RT-PCR方法。因此，所开发的高度灵敏且特异的HCV抗原检测方法可应用于HCV感染的大规模筛查。
