Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method.
BACKGROUND
Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR)-based assays and enzyme-linked immunosorbent assays (ELISA) are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS) method was applied to detect HBV and HCV antibodies rapidly and simultaneously.


METHODS
Chemically modified glass slides were used as solid supports (named chip), on which several antigens, including HBsAg, HBeAg, HBcAg and HCVAg (a mixture of NS3, NS5 and core antigens) were immobilized respectively. Colloidal nano-gold labelled staphylococcal protein A (SPA) was used as an indicator and immunogold silver staining enhancement technique was applied to amplify the detection signals, producing black image on array spots, which were visible with naked eyes. To determine the detection limit of the protein chip assay, a set of model arrays in which human IgG was spotted were structured and the model arrays were incubated with different concentrations of anti-IgG. A total of 305 serum samples previously characterized with commercial ELISA were divided into 4 groups and tested in this assay.


RESULTS
We prepared mono-dispersed, spherical nano-gold particles with an average diameter of 15 +/- 2 nm. Colloidal nano-gold-SPA particles observed by TEM were well-distributed, maintaining uniform and stable. The optimum silver enhancement time ranged from 8 to 12 minutes. In our assay, the protein chips could detect serum antibodies against HBsAg, HBeAg, HBcAg and HCVAg with the absence of the cross reaction. In the model arrays, the anti-IgG as low as 3 ng/ml could be detected. The data for comparing the protein chip assay with ELISA indicated that no distinct difference (P > 0.05) existed between the results determined by our assay and ELISA respectively.


CONCLUSIONS
Results showed that our assay can be applied with serology for the detection of HBV and HCV antibodies rapidly and simultaneously in clinical detection.
Department of Biotechnology, College of Life Sciences, Wuhan University, 430072, Wuhan, Hubei, People's Republic of China. dll000212@yahoo.com.cn
Lianlian
L
Duan
Yefu
Y
Wang
Shawn Shun-cheng
SS
Li
Zhixiang
Z
Wan
Jianxin
J
Zhai
D016174:Hepacivirus / Q000276:immunology*; D006510:Hepatitis B Antibodies / Q000097:blood*; D006512:Hepatitis B Core Antigens / Q000276:immunology; D006514:Hepatitis B Surface Antigens / Q000276:immunology; D006513:Hepatitis B e Antigens / Q000276:immunology; D006515:Hepatitis B virus / Q000276:immunology*; D018937:Hepatitis C Antibodies / Q000097:blood*; D018936:Hepatitis C Antigens / Q000276:immunology; D006801:Humans; D036103:Nanotechnology; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D016622:Silver Staining
D006510:Hepatitis B Antibodies; D006512:Hepatitis B Core Antigens; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D018937:Hepatitis C Antibodies; D018936:Hepatitis C Antigens