Production And Preliminary Application Of A Visual Protein Chip To Differentiate Antibodies Of Four Poultry Diseases

Biochip technology, which was developed along with the human genome project (HCG), is a new technique and method applied in life science research. As the gene chip technology has gradually become mature and many outstanding achievements have been obtained, the use of protein chip technology emerged relatively recently. Protein chip technology has shown its great potential in the functional analysis of the proteome, disease diagnosis and drug development.To produce a visual protein chip that can detect antibodies against avian influenza virus, newcastle disease virus, infectious bronchitis virus and infectious bursal disease virus simultaneously. The establishment of protein chip include the selection of suitable substrate, the delivery of capture molecular, the hybridization of antibody and the removal of unspecific binding.Purify the 4 viruses protein with Ultracentrifugation and sucrose density gradient centrifugalization method ,the proper concentration purified protein were spotted on a aldehyde group modified glass slides respectively to generate protein chips. Positive serum against these viruses were hybridized to these antigen, then the bound antibodies were detected with nano-gold labelled second antibody and developed with silver staining method, a platform of a visual protein chip to differentiate 4 kinds of avian diseases was constructed.; Furthermore, we have studied specificity and the correlation of intensity valuabel with the concentration of antibodies of this visual protein chip, then the visual protein chip was used to test 18 specimen .After the condition was optimized, relative antigen could be hybridized specifically with the probe of this protein chip and strong signal was showed. Intensity valuabel showed linear reference in the concentration range of 50μg/mL～300μg/mL of antibodies. This protein chip showed good sensibility compared with traditional method. Through a series of optimization experiments, an optical protein chip for differentiating antibodies of four poultry diseases was constructed. This chip is characterized by rapidity, simplicity, high sensitivity, good specificity, no need for special instrumentation and low cost. The detection reliability of serum specimens is high and the sensitivity is also higher than that of the agar diffusion reaction, and it could become a popular application.This method, which combines the immune colloidal gold tech(?)ique, the silver developing technique and protein chip technique, selects purified totivirus protein as the captured antigen for the first time, and is an innovation and breakthrough in serum detection techniques. Our visual protein chip will play a considerable role in promoting the popularization of protein chip technology; this technology lays a good foundation for further construction of integrated chips of high density for detecting multiple antibodies.