Establishment Of A Competitive ELISA (cELISA) For Detection Of AIV Antibodies

Avian Influenza (AI) is one of fatal infectious disease caused by influenza A virus in avian. Itfirst appeared in Italy more than 100 years ago(around 1878), and so far, different strains werefound and caused important economic losses in the avian industry throughout the world.Accordingto the difference of HA gene,which is separated 15 stype from H1 to H15;according to difference ofNA gene,which is separated 9 stype from N1 to N9.It is no cross-reaction between different HA orNA,so it is diffcult to detective.M1 and NP are the type-specific protein of the AIV, and they are prevalent to detect antigensbecause of conservative gene order among different subtypes. This research is aimed to establish-ment of a competitive ELISA for detection of AI in order to provide technique to for epartdiagnoses of AI. We establiashed the M1-cELISA and NP-cELISA .Recombinant encoding the M1and NP of avian influenza virus (AIV) was generated and the appropriate protein was expressed inBL21(DE3) and Rosetta(DE3). Purified recombinant M1 and NP and the the sera ofrabbit-anti-M1, rabbit-anti-NP were used to establish a competitive ELISA (cELISA) for thedetection of antibodies in sera of all of poultry. The cELISA allows for a rapid serological andspecies independent diagnosis of AIV infection. Tests to evaluate this method were carried outusing sera of ducks,gooses,chicken,and poultry field sera, which tested in the haemagglutinationinhibition (HI) assay, and several poultry species infected with other viruses.Results showed: 22.85% and 17.49% was therefore assessed as the preliminary cut-off value ofM1-cELISA and NP-cELISA. They were negative on detecting chicken anti-NDV sera, anti-IBVsera, anti-IBDV sera,anti-ILTV sera, anti-MDV sera, anti-APV sera. Tests carried out using sera ofduck chicken,goose and some wild birds revealed widely corresponding results obtained by HIassay and cELISA indicating that this test is applicable for flock diagnosis. Differing results wereobtained for individual samples. Their inner and outer plate coefficient of differentiation were 4.3%and 8.6%, 3.6% and 9.1%, respectively. The evaluation of the test demonstrated a high sensitivityand specificity of NP-ELSIA and M1-ELISA for the type A Influenza detection.M1-cELISA and NP-cELISA for type A Avian Influenza detection could be applied to thedetection and monitoring for evaluation of AI immunizing efficiency. Therefore, promote thedevelopment of the culturation farm in our country directly. They could also be applied toserological monitoring, and this is significant for our understanding of AI infections history of AIV,the ecological distribution of AIV and the prevention, prewarning and precaution of AI all over theworld. Meanwhile, they are the technological storage for distinguishing AI, between immunized and infected AIV.