Preparation Of Monoclonal Antibodies Against The Haemaglutinin In Aivan Influenza Virus Subtype H5,H9 And Development Of Sandwich ELISA Differentiability Kit For Detecting AIV Antigen

Avian Influenza (AI) is one of syndrome disease of birds caused by influenza A virus in avian. At present, highly pathogenic avian influenza (HPAI) is listed in A infectious diseases by World Organization for Animal Health (OIE). Since AIV was found in Italy 1878, there were breaking out and prevalence caused by different strains of AIV and caused important economic losses in the avian industry throughout the world. Especially, events that AI infected human being happened in south-eastern Asian (2003 2004), which shocked mankind and caused our attention to commonality sanitation of AI. Because of broad area, large quantity of birds, frequent circulation of poultry product, it is difficult to remove completely from the external environment after once the bird flu happened, The antigenic drift and antigenic shift of AIV makes it be possible that low virulence influenza shift to HPAI, and which makes potential menace to the avian industry in our country. It is importance to diagnose timely and speedily in order to win golden time for forepart prevention and therapy, and to reduce economic losses furthest. This research is aimed to develop ELISA for detection of AIV in order to provide technique to diagnoses of AI in short time, especially to differentiability diagnoses of AIV H5 and AIV H9. The results of research are summarized as following:1. Preparation of monocloneantibody(McAb) of anti-haemaglutinin of AIV H5 and AIV H9AIV H9 and AIV H9 were inoculated separately into 9 to 10day embryonating chicken eggs separately by the allantoic route, then allantoic fluid was harvested and centrifuged with 27,000r/min, and deposit was redissolved by 0.01mol/L PBS (pH7.2) which is 1/30 volume of allantoic fluid. Immune spleen cells which get from the Balb/c mouse immunized with redissolved fluid of AIV H5 and AIV H9 and Sp2/0 myeloma cells which disfigured hypoxanthine-guanine phosphoribosyl transferase (HGPRT) were amalgamated, and seven strains of hybridized cells were obtained after being cloned. The number of chromosome of those seven strains hybridized cells is all between82 to 92, which is more than that of their parents. The cells could secrete antibody stably after being froze and anabiosis. The HI titers of ascites fluids to corresponding subtype AIV are 2¹³-2²⁰. No cross reaction of HI be found to McAb H52B4,H53C9 and H55E8 with AIV H9, EDS_-76V, NDV, IBV, ILTV, APV, MDV and IBDV, and to McAb H91C6, H91G3, H92C2 and H94C4 with AIV H5 and other virus. The appetence is best between McAb H52B4 and AIV H5, and between McAb H94C4 and AIV H9. Obtainment of the monocloneantibodies provided the foundation for the development of enzyme-linkedimmunosorbent assay (EL1SA) for AIV H5 and AIV H9.2. Development of enzyme-linked immunosorbent assay (ELISA) for AIV H5 andAIVH9.A sandwich ELISA for detection of AIV H5 antigen was developed by using monocloneantibody (McAb) of anti-haemaglutinin of AIV H5 as the first antibody coated on the ELISA plate and rabbit anti-AIV IgG regarded as the second antibody. And another sandwich ELISA for detection of AIV H9 antigen was developed by using purified chicken anti-AIV IgG as the first antibody coated on the ELISA plate and monocloneantibody (McAb) of anti-haemaglutinin of AIV H9 regarded as the second antibody. The dilution titers of positive AIV H5, and AIV H9 sample by corresponded ELISA was 16 times higher than that by hemagglutination test Positive AIV H9 sample can not be detect by ELISA for detection of AIV H5 antigen, and positive AIV H5 sample can not be detect by ELISA for detection of AIV H9 antigen. No cross reaction was observed with other viruses of birds (Newcastle disease virus (NDV), infectious bronchitis virus (IBV), eggs drop syndrome virus (EDS-76V), infectious laryngotracheitis virus (ILTV), infectious bursal disease virus (IBDV), avian pox virus (APV) and Marek's disease virus(MDV) )by both the ways. The results illuminated that tow methods have highly specificity and sensitivity. A Sandwich Enzyme-linked immunosobent assay test Differentiability kit based on previous work was developed for detection of avian influenza virus (AIV) antigen including all AIV subtypes detecting, subtype of ATV H5 and subtype of AIV H9. The kit consists of twelve reagents and a piece of 96 well microtiter plate. It is highly specificity, sensitivity and repeatability to detecting AIV antigen with the test kit. It was stable well when storing at below -10°C for tow months up to the present. It is very easy to manipulate, convenient to schlep and the result can be reported speedily and veraciously.