Exploratory Development Of Fast Antidiastole Test Paper Differentiating Infectedfrom Vaccinated Poultry For Avian Influenza

Now as to the problems which happened in the detection on AIV, with the established fast antidiastole method to differentiate field infection from vaccination in avian infectious disease, by the optimization of various components and experiment condition, take a further step, fast antidiastole kit on AIV infection could be assembled and installed, large scale research on the serum of artificial infection broiler and clinical serum, the concrete achievements are as follows:1. Bolting of expressive qualification in E.coli of non-structural protein (NS1) of AIV and the optimization of various reaction conditionThe best expressive quantity of Pkg-NS1 should be derivned for 6 hours at 28℃. Results of Western-blotting showed that GST-NS1 could react to the chicken antiserum against AIV with high immunological competence. Highly purified expressive protein that the density is 1.38 mg/mL could be generated by purified with GST label protein by the method of affinity purification. The optimum condition was established that the ideal pH is 8.0 The ideal concentration is 10|ig monoclonal antibody/mL colloidal gold during the labeled reaction.2. Advanced development on fast antidiastole test kit on infection of avain influenza virusBasing on the successful fast antidiastole test paper, the fast antidiastole kit on infection of avian influenza virus had been assembled and installed. The test on keeping time of kit indicates: There is no evident difference on the specificity and sensitivity after the kit has been kept for 4 month at 4℃; the same is ture while kept for 2 month at room temperature. In conclusion, the kit has been proved that the minimum period of validity is 4 month at 4℃.3. Research on the regularity of NS antibody of generation, growth and decline after artificial infected by AIVAt third day after infected, and the positive rate up to 89% at 11 days then declined rapidly, at 21 days, 10% detection rate lowered,at last, disappeared until 26 days post-infection, whilst the vaccinated-chickens and PBS-control chickens were negative in NS1-Abs detection. The positive rate of NS1-Abs was strongly compatible to the rate morbidity and mortality of the infected broilers. Though the detection on NS1 antibody are both negative between the control group and the immuned group with inactivated vaccine, the rise and fall regularity of HI-Ab of both group are basically consistent. By the experimental infection tests on broilers, the situation of the antibodies against NS1 of AIV were first understood and discovered, furthermore it testified that the immunochromatographic strip of detecting NS1-Ab was not only feasible to identified rapid and earlier whether the respiratory infecting chickens infected AIV or not, but also could distinguish AIV-infected-chicken from vaccinated-chicken.4. Exploratory development of fast antidiastole test kit on AIVMethod through the fast-detection to 940 clinical sera of immunizing fowl from 21 henneries of Hubei province and Henan province, compared with the orthodox methods such as hemagglutination inhibition test (HI), the results are as follows, the positive detection rate is 18.8% by the method of GICA test, the detection rate of subtype H5 and subtype H9 of AIV by the method of HI test in turn are 73.4% and 30.9%. In addition, the positive samples of subtype H5 or subtype H9 of GICA test is still positive in the HI test. By above knowable, the mixed infection (H5 and H9) and the infection after immuned are both existed domesticly.The fast antidiastole kit on infection of AIV with many advantages that such as simple and rapid manipulation, easily analyzed, and it doesn't need professional operater. especially suitable for the primary detecion which without specification and expensive equipments.