The Correlative Comparison Of Testing Infectious Bronchitis Antibody By HI And ELISA

In the tests,infectious bronchitis virus(IBV)inoculate 10- or 11-day-old specific pathogen free embroyonated chicken eggs and incubation for 36 hours at 37℃. Allanto-amnionic fluid (AAF) was harvested from chilled viable embryos and clarified by centrifugation at 1,000×g for 30 minutes, the pellet was discarded, and virus was concentrated from the supernatant fluid by centrifugation at 39,000×g for 50 minutes, or concentrated by PEG8000. The virus-containing pellet was resuspended to 1/100 the original AAF volume with HEPES-buffered saline. The concentrated virus was treated with the trypsin, by mixing equal parts of the enzyme(1unit/ml) and virus concentrate and incubating the virus-enzyme mixture at 37℃for 3 hours. The concentrated virus was treated with the enzyme phospholipaseC(Type-1), neuraminidase in the same method, the terminal concentration of PLC1 and neuraminidase both are 2.5units/ml, The mixture was agitated occasionally during incubation. The enzyme-treated virus concentrate- resultant IBV HI antigen, was tested for HA activity and stored at 4℃.IBV HI antigen tested demonstrated positive HA activity with chicken red blood cells. But in IBV HI test identified the antigen that treated by trypsin was not specifically inhibited by immune serum,antigens that treated by PLC1 or neuraminidase were specifically inhibited by immune serum.The self made antigen compare with the imported antigen, both antigens tested demonstrated high HA titer.The test compared and investigated each elements that effected the IBV HI test, selected 1%CRBC, pH6.5 0.01M HEPES-buffered saline as diluent, antigen- antibody reaction at room temperature for 30min, after addition of RBCs, the plate was inbubated at 4℃, and the test was read after 50 minutes of incubation, etc., establishment of the HI test for detection of IBV HI antibody. The test had strong specificity and repeatability.Detecting 25 IB unimmune chicken serum using the established IBV HI test, and challenge test, determined the immune critical line is 6log2. Comparing the detection results of IBV HI test and ELISA kit for detecting IBV M41 antibody, when the HI titer was greater than 6log2, the S/P was greater than 0.2; when the HI titer was less than 6log2, the S/P was less than 0.2. The HI test showed significant correlations with ELISA, and had strong specificity, repeatability and sensitivity. Selecting 92 IBV unknow serum with randomly, detected them using HI test and ELISA, the coincidence rate of detection result of HI test and ELISA is 92.4%.Study showed that the establishment IBV HI test that using self made IBV HI antigen was simple, fast and accurate, can be used as a scientific and practical method for IB diagnosis, immune effect detection, and epidemiology investigation and research.