Research And Application Of A Visual Protein Chip For Diagnosis The Antibodies Of Avian Influenza, Infectious Laryngotracheitis And Infectious Bursal

Purify the 3 viruses protein with Ultracentrifugation and sucrose density gradient eentrifugalization 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 3 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 technique, 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.The visual diagnostic protein chip platform is established by using recombinant NS1 protein as antigen.In the experiment, the expression of NS1 protein was diluted to 3000μg/ml.Known AI infections serum and immune sera each 25, SPF chickens serum, ND positive serum and IBD positive serum each 10 were detected by using protein chips, the test results were 100% in accordance by infected serum and immune serum, in dicating that the method has a good accuracy. SPF chicken serum, ND positive serum, IBD positive serum test hasn't nonspecific reaction, indicating a good specificity of protein chip. Infected serum collected at different time points were detected by using protein chip, would detected on day 8 of infection,but detected by agar gel precipitin test (AGP) in the first 14 days of infection. Obviously, protein-chip detection is more sensitive than AGP method. Established diagnostic test platform for protein chip can quickly and accurately divide the nature infection and vaccination immune poultry, the test results can be determined by the naked eye alone, be helpful of using at any moments, which is the new breakthroughs for controlling and purification technology of avian influenza.