| Background:Avian influenza is one of the avian drastic infectious diseases caused by the influenza viruses A. In China, the fowl are infected mostly by H5 and H9 subtype. H5 has high pathogenicity and spreads quickly; it also has short course, high virulence and fatal to the fowl. H9 has pathogenicity too. The occurrence of AI has been more frequency, more widely and repeated relapse for recent years, and it has caused huge economic damage and influenced the society badly all over the world. Otherwise, more and more evidences had indicated that the AIV could breakthrough the barrier in different genera and could threaten the human lives acutely. Therefore, scientists have been paid more attention to the AI's rapid diagnosis, prevention and cure.Piezoelectric immunosensor is the most popular issue in the field of biosensors combining with the high mass sensitivity of the quartz crystal sensor and the high specificity of the immuno-reaction. Compared with the traditional immunoassay, this kind of sensor has many advantages such as simple instruments, high sensitivity, quick response, free label, easy operation, low price, open country available, etc. At present it has been widely studied in the fields such as clinical diagnosis and injurant detection.Now the detecting technology of AIV is tedious operation and cost lots of time. Hence, there are momentous theoretical and practical significances to use corresponding AIV subtype monoclonal antibodies as biotic sensitive materials to fabricate a piezoelectric immunosensor for AIV detection.Objectives:To develop an AIV piezoelectric immunosensor with different immobilization of antibody as biotic sensitive material; and explore the feasibility of this kind of immunosensor applied in rapid detection of AIV.Methods:1. H5 and H9 subtype AIV monoclonal antibodies are deposited on the gold electrode's surface of 10 MHz quartz AT-cut, 5mm in diameter crystal by three different immobilization of antibody (polyethyleneimine adhesion and glutaraldehyde cross-linking method,SPA method and self-assembly method) to fabricate the piezoelectric immunosensor. 2. Compare the merits and shortages of the three methods, and find the suitable immobilizing method and conditions.3. Compare the frequency shifts of the immunosensors fabricated by different antibody immobilization in detecting H5 and H9 antigens.4. The polyethyleneimine adhesion and glutaraldehyde cross-linking method (PEI method) was selected to fabricate the AIV piezoelectric immunosensor, which repeatability, linear range, sensitivity and cross-reaction were studied and compared with chick embryo separation.Results:1. PEI method: the optimum condition of the immobility of H5 and H9 monoclonal antibodies are both 1:10 dilution, and it has better response to antigens.2. SPA method: the optimum conditions of the immobility of H5 and H9 monoclonal antibodies are both 1:5 dilution. It wouldn't detect the corresponding antigens yet if the antibodies had been immobilized to the gold electrode; the qualitative detection could be gotten by combining spa quartz crystal with antigen-antibody compounds.3. Self-assembly method: the optimum conditions of the immobility of H5 and H9 monoclonal antibodies are both 1:10 dilution. But it has little response to antigens.4. Comparison to the immobilization of antibody in three methods: the immobilization of antibody by self-assembly method is better than the other two.5. The H5 AIV piezoelectric immunosensor fabricated by PEI method: the CV of H5 antigen is 14%, linear range is between 1:2～1:40, correlation coefficient is 0.9894, and there is no cross-reaction to H9 antigen. The H9 AIV piezoelectric immunosensor: the CV of H9 antigen is 21%, linear range is between 1:10～1:160, correlation coefficient is 0.9832, and there is no cross-reaction to H5 antigen. Coincidence is 91.7% compared with chick embryo separation.Conclusion:H5 and H9 subtype AIV monoclonal antibodies are deposited on the gold electrode's surface as biotic sensitive materials, the PEI method is the best one for fabricate a piezoelectric immunosensor, which sensitivity, specificity, repeatability and linear range can reach the detection standards, and could be used to the rapid detection of AIV.
|
PDF Full Text Request