Development And Application Of The Colloidal Gold Assays For Detection Of Swine Influenza Virus

Swine influenza virus infections are common causes of respiratory disease in pigs. The majority of the influenza viruses circulating in pigs are HI and H3subtypes. The pandemic H1N12009virus has been isolated from pigs in many countries, including China, the USA and Canada. So far, Evidence of pig to human influenza virus transmission was found in farm workers who showed increased prevalence of elevated antibody titres to both swH1N1and swH1N2. Therefore, it is critical to have access to assays swine flu employing rapid, reliable and affordable technology, to provide technical support for the control and depuration of swine flu virus. Influenza virus has a high degree of variability, even within the same subtype virus, HA protein variation is great. It is difficult to filter to a monoclonal antibody specific identification of all strains of the same subclass. Screened through joint complementary roles specific identification of all strains of the same subtype monoclonal antibody is also an important way for preparation of high-sensitivity detection kit. In this study, two swine influenza virus colloidal gold detection kit was developed using monoclonal antibody technology, the results were reported as follows.1. Preparation of monoclonal antibodies of the swine influenza virus H3subtype.In this study, through existing laboratory and NCBI indexed between1990-2011swine influenza virus subtype H3HA protein sequence evolution analysis, scree ned two swine influenza virus virus H3subtype, GD3and HN3. GD3and HN3was inoculated proliferation virus in eggs, lus formaldehyde inactivated allantoic fl uid harvested after72h. Sucrose gradient centrifugation immunize Balb/c mice, tak e immune mouse spleen cells with SP2/0bone marrow tumor cell fusion. HI and ELISA screening, after3times clones were screened to15anti-swine influenza vir us H3subtype monoclonal antibody. Measured by the test, the15mAb only react with the swine influenza virus subtype H3and not react with the other subtypes of influenza virus, and with good specificity.2. Development of swine influenza virus H3subtype colloidal gold detection kit.Screened4monoclonal antibody from15monoclonal antibody against swine influenza virus subtype H3, in which two mixed spraying film, two mixed labled gold, developed wide identification of swine influenza virus subtype H3colloidal gold detection kit. This kit detects the52pigs clinical nasal swabs and200lung samples,9positive nasal swabs and7positive lung samples were detected, virus isolation of test sample to prove that no undetected, the specificity of of the kit is100%.Other subtypes of influenza virus detection using this kit, such as:H1subtype, H5subtype, H9subtype, H10subtype of influenza virus, the results were negative. And with this kit to detect pathogens that cause common pig virus disease, such as:PRRSV, PRV, PCV2, CSFV, results were negative. This kit has a specificity, sensitivity, high stability and can batch determination, provided new avenues for clinical detection of swine influenza virus H3subtype.3. Development of the swine flu virus H3subtype and pandemic influenza A virus H1N1(H1N1pdm) bivalent colloidal gold detection kitWith laboratory prepared and save anti-pandemic H1N12009monoclonal antibody2B3and anti-swine influenza virus H3subtype monoclonal antibody prepared in this study, established bivalent colloidal gold assay kit of swine influenza virus subtype H3and pandemic H1N12009. This kit test line as the detection of swine influenza virus H3subtype1F6and3D1spray film as the detection of the swine flu virus H3subtype and pandemic influenza A virus H1N1(H1N1pdm) line2B3spraying film, film sprayed with goat anti-mouse polyclonal antibody as a control line, to the gold labeled2B8, the gold labeled4H9and the gold labeled2B3coated conjugate pad. The bivalent colloidal gold assay kit of swine influenza virus subtype H3and pandemic H1N12009is specificity, high sensitivity and high stability. This kit detects H3subtype virus, pandemic H1N12009influenza virus as positive reaction, detect classical H1N1swine influenza virus, H5subtype of influenza A virus, H9subtype influenza virus, H10subtype influenza virus, PRRSV, PRV, PCV2, CSFV, showed negative reaction. This bivalent colloidal gold test kit is important for the diagnosis, prevention, control and monitoring of swine flu virus and H1N1pdm, and also laid a solid foundation for the research and development of multi-linked colloidal gold test kit.