Zhang Wen, Huang Jian, Zhou Mei-Fen, Chen Li-Yan, Ding Ya-Ping, Cao Heng-Jie, Geng Yong-Yao, Wang Sheng-Qi
Institute of Radiation Medicine, Beijing, China.
Mol Diagn. 2005;9(2):81-7. doi: 10.1007/BF03260075.
As a contagious disease caused by hepatitis C virus (HCV) hepatitis C is a serious threat to human health. Therefore, the detection and verification of HCV infection is very important in the treatment of hepatitis C. This study investigated the preparation, quality control, and clinical evaluation of a protein chip capable of simultaneously detecting different HCV antibodies. The aim was to establish a convenient method for the detection of HCV.
To prepare the protein chip, six antigens including five recombinant HCV antigens (chimeric, core, NS3, NS4, and NS5) and interleukin (IL)-1 were arrayed onto aldehyde-coated slides and blocked using 10% calf serum in phosphate buffered saline. After dilution with sample solution, the serum sample was added to a reaction well on the protein chip. After incubation for 30 minutes at 37 degrees C, fluorescence Cy3-labeled rabbit antihuman IgG was added and incubated again for 30 minutes at 37 degrees C, and then scanned. Positive or negative controls were established from serum samples with or without HCV infection. Clinical evaluation was done by detecting 490 serum samples using the protein chips and ELISA reagents, with 150 of the 490 serum samples confirmed by recombinant immunoblot assay (RIBA).
The protein chip for detection of five HCV antibodies was successfully prepared. Fifteen positive controls and 15 negative controls were established as standard samples for quality control. The quality control-passed protein chip was tested again using the standard of the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP), and met the quality control criteria prescribed by the NICPBP. In the clinical evaluation with 490 samples, the coincidence rates between the protein-chip assay and ELISA were 97.4% for positive and 100% for negative results. Five inconsistent samples that were positive in ELISA, but non-positive (four samples) or negative (one) in the protein-chip assay, were confirmed by RIBA (gold standard) to be four non-positive and one negative. The results of 150 samples showed the coincidence rates between protein chip and RIBA were 98.15% for positive and 96.88% for single-segment positive.
The protein-chip assay has higher sensitivity and specificity than ELISA and has a high coincidence rate with RIBA. The protein chip, characterized by its easy operation and low economic cost, will be very useful for in vitro detection of HCV antibodies.
丙型肝炎是由丙型肝炎病毒（HCV）引起的一种传染病，对人类健康构成严重威胁。因此，丙型肝炎病毒感染的检测与验证在丙型肝炎治疗中非常重要。本研究调查了一种能够同时检测不同丙型肝炎病毒抗体的蛋白质芯片的制备、质量控制及临床评估。目的是建立一种便捷的丙型肝炎病毒检测方法。
为制备蛋白质芯片，将包括五种重组丙型肝炎病毒抗原（嵌合、核心、NS3、NS4和NS5）及白细胞介素（IL）-1在内的六种抗原排列在醛基包被的载玻片上，并用磷酸盐缓冲盐水中的10%小牛血清封闭。用样品溶液稀释后，将血清样品加入蛋白质芯片上的反应孔中。于37℃孵育30分钟后，加入荧光Cy3标记的兔抗人IgG，再次于37℃孵育30分钟，然后进行扫描。通过有或无丙型肝炎病毒感染的血清样品建立阳性或阴性对照。通过使用蛋白质芯片和酶联免疫吸附测定（ELISA）试剂检测490份血清样品进行临床评估，490份血清样品中的150份通过重组免疫印迹法（RIBA）进行确认。
成功制备了用于检测五种丙型肝炎病毒抗体的蛋白质芯片。建立了15份阳性对照和15份阴性对照作为质量控制的标准样品。通过中国食品药品检定研究院（NICPBP）的标准对质量控制合格的蛋白质芯片再次进行检测，符合NICPBP规定的质量控制标准。在对490份样品的临床评估中，蛋白质芯片检测与ELISA检测的阳性符合率为97.4%，阴性符合率为100%。ELISA检测呈阳性但蛋白质芯片检测为非阳性（4份样品）或阴性（1份样品）的5份不一致样品，经RIBA（金标准）确认为4份非阳性和1份阴性。150份样品的结果显示，蛋白质芯片与RIBA的阳性符合率为98.15%，单片段阳性符合率为96.88%。
蛋白质芯片检测比ELISA具有更高的灵敏度和特异性，与RIBA的符合率高。该蛋白质芯片操作简便、经济成本低，对丙型肝炎病毒抗体的体外检测非常有用。
