Research On The Visual Protein Chip And Its Relevant Techniques

Protein chips have emerged as an exciting technology for the broad characterization of the activities and interactions of proteins. In the past few years, protein chip and microarray technology has shown its great potential in the functional analysis of the proteome, clinical diagnostics, drug screening and testing, and disease monitoring. It allows fast, easy and parallel detection of thousands of addressable elements in a single assay. For instance, the potential of this technology to diagnose human diseases, such as leukemia, breast cancer and, potentially, heart failure, has stimulated much interest.Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. A variety of HBV and HCV markers have been used to detect HBV and HCV infection. Gene amplification tests, such as PCR-based assays are used to diagnose and monitor the efficacy of treatment. However, these methods require cumbersome procedures and expensive equipment, thus requiring considerable skills and high costs. Immunoassays are generally easy and inexpensive. So far, some immunological methods such as enzyme-linked immunosorbent assays (ELISA) and rapid diagnostic paper have been used in clinical practice. While the value and significance of these methods are beyond argument, they suffer from several disadvantages, mainly their inability to produce results simultaneously. In our assay, we established a platform on which protein chips with a high sensitive visual detection based on Nano-gold Immunological Amplification and Silver Staining (NIASS) method was applied to detect HBV and HCV antibodies rapidly and simultaneously.In our assay, 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. The results were analyzed with paired chi-square test and validated by parameters including validity, sensitivity, specificity, crude agreement, adjusted agreement, youden's index and predictive value.The results showed that 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. All the results showed that our assay can be applied with serology for the detection of HBV and HCV antibodies rapidly and simultaneously in clinical detection.