Development And Application Of Assays For Detection Of Subtype A/H1 Influenza Virus

March 2009, the "swine flu" epidemic outbreak in Mexico, and quickly spread worldwide. Through research, it is affirmed that the epidemic situation is caused by a new type of H1N1 virus. Humans do not have natural immunity to the virus.So, H1N1 virus is spreading around the globe. According to the latest data of World Health Organization, there are 214 countries and regions in the world reporting the confirmed cases of influenza A and 18449 death cases. A/H1N1 influenza caused huge impacts on the health of animal and people and also led to huge economic loss. For the huge threats to animal husbandry and human public health and safety caused by A/H1N1 influenza, we shall carry out monitoring on antigen of influenza A regularly to understand the prevalence situation of influenza A virus timely, in current market without reagent kit to detect A/H1 subtype pig influenza virus (North America influenza) and the blank without identification diagnosis reagent kit to discriminate A/H1 subtype pig influenza virus (North America influenza) and classical H1 subtype influenza virus. So, it is particularly important to establish a series of simple, rapid, specially sensitive and visual and effective monitoring method for influenza A virus. In the research process of the subject, monoclonal antibodies of many anti-influenza A H1 subtype influenza virus hemagglutinin are prepared and with the antibody as raw materials, A/H1N1 subtype influenza virus double-antibody sandwich ELISA detection method and A/H1 subtype influenza virus immune colloidal gold technique are established respectively and they have the advantages of high sensitivity, strong specificity, simple and convenient operation and batch detection, which lay the physical basis for further diagnosis and monitoring of A/H1 subtype influenza antigen.1. Preparation of A/H1 subtype influenza hemagglutinin monoclonal antibodyA/H1 subtype of influenza virus was cultured in chicken embryo. The obtained virus was inactivated with formaldehyde. Balb/c mice were immuned with the purified A/H1 influenza virus antigens. Splenocytes from the immunized mice were fused with SP2/0-Agl4 myeloma cells, and positive hybridoma clones were screened by hemagglutination inhibition (HI) and limited dilution method was performed to subclone the positive clones. After three cycles of subcloning, ten McAbs against the hemagglutinin of A/H1 influenza virus hemagglutination were obtained, get the Specificity and titer monoclonal antibody, designated as 1C9,2B3,2C5,2H7,3G12,4D7,4B12,4E1,5D5and 5F7. The HI title of ascites were 213～219. Determination with test,7 McAbs could only occurred hemagglutination inhibition reaction with the A/H1 subtype of influenza virus, but do not occurred hemagglutination inhibition reaction with the classic H1 subtype of swine influenza viruses and other subtypes of influenza virus, this 7 McAbs with good specificity, and the remaining 3 McAbs not only occurred hemagglutination inhibition reaction with the A/H1 subtype of influenza virus but also with some classic H1 subtype of swine influenza virus, but other subtypes of influenza virus does not cross reaction.2. Establishment of A/H1 subtype influenza virus detection methodBased on anti-A/H1 subtype influenza virus hemagglutinin monoclonal antibody, establish A/H1 subtype influenza virus double-antibody sandwich ELIS A detection method and A/H1 subtype influenza virus immune colloidal gold technique respectively and they have the advantages of high sensitivity, strong specificity, simple and convenient operation and batch detection. These two methods are used to detect other subtype influenza virus, such as classical H1 subtype, H3 subtype, H5 subtype and H9 subtype influenza virus and the detection result is negative. What is more, these two methods can also be used to detect major virus of livestock and poultry, such as, CSFV, PRRSV, PRV, JEV, NDV, IBDV, EDSV and the detection result is negative. All above detections show that these two methods have good specificity. A/H1 subtype influenza virus double-antibody sandwich ELIS A detection-based A/H1 subtype influenza virus double-antibody sandwich ELIS A reagent kit makes up the gap in current market without reagent kit to detect A/H1 subtype pig influenza virus (North America influenza) and the blank without identification diagnosis reagent kit to discriminate A/H1 subtype pig influenza virus (North America influenza) and classical H1 subtype influenza virus. The above detection methods for A/H1 subtype influenza virus can rapidly and accurately detect A/H1 subtype influenza virus in animal group and human groups. Therefore, these two methods have extremely important significance to assessment, prevention and epidemiology survey of A/H1 subtype influenza virus.