Development Of Monoclonal Antibodies Against H1n1Influenza Virus Of Pig Origin And Double Antibodies Sandwich Elisa For H1n1Influenza Virus Detection

Swine influenza (SI) is a highly contagious respiratory disease caused by swine influenza virus(SIV), including H1N1, H1N2, H3N2sutypes of SIV. Swines are the host of both avian origin and human origin influenza viruses, and can act as an animal reservoir where influenza virus genes become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses. The pandemic of SIV may cause economic losses, and it is very important to public health significance. Four hundred and thirty-three nasopharyngeal swabs of swine were collected in a slaughter house of Yangzhou in Jiangsu Province in2011. Six swine influenza virus were isolated and identified to be SIV by hemagglutinin inhibition (HI) and RT-PCR assays.6-week-old female BALB/c mice were immunized with H1N1strain isolated in Guangdong in2009, and then collected the spleen cells and fused with Sp2/0myeloma cells. Hybridoma cells were screened by HI, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IFA) test. The positive clones were subjected to limited dilution for subcloning. Eight mAbs were developed after several subcloning and named as2B11,1B4,2C7,3A4,6A9,6B2,3B8,6D11respectively. The8monoclonal antibodies (McAb) were against to different protains. McAb2B11,6D11was against hemagglutinin (HA); McAbs1B4,2C7,6A9were against neuraminidase (NA); McAb3A4,6B2,3B8was against matrix proteins (M1). Five of the eight McAbs (2B11,1B4,2C7,6A9,6D11) were specific to H1N1SIV, and not reacted with H5, H9subtype avian influenza virus (AIV), Newcastle disease virus (NDV), while McAb3A4,6B2,3B8reacted with all the types of A influenza viruses we tested by IFA. The fluorescence of McAb3A4,6B2,3B8reacted with MDCK-90cells infected with SIVs was located in the cytoplasm, while those of the other five McAbs’were located around the cell membrane.Accoding to the characteristics of8McAbs,3A4and1B4were chosen for development of Double antibodies sandwich ELISA (DAS-ELISA). After two McAbs were purified by octanoic acid and chromatography,3A4were labeled with horseradish peroxidase as detecting antibody,1B4was chosen as coating antibody. The DAS-ELISA has high sensitivity, and no cross-reaction was found when AIV and NDV were tested by this assay.50nasopharyngeal swabs,20lung tissues and20hilar lymph nodes were tested by the DAS-ELISA method and the virus isolation in parallel. The results showed that coincidence rate between the DAS-ELISA method and the virus isolation was97.8%. The DAS-ELISA method is simple, fast and sensitive in the detection of H1N1SIV, and can be used generally in clinical.