Preparation Of Monoclonal Antibodies Of Swine Influenza Virus H1N1and Development Of Colloidal Gold Strip For Detection Of The Influenza Virus

Subtype H1N1of swine influenza virus (SIV) were inoculated into9-11-day-old chickenembryos, dose for100ul/pieces, and incubated for72hours. HA assays were performed todetect that the HA titer was1:256. BALB/C mice were immunized with4%formaldehydeinactivated virus and inactivated150μl each, then they were boosted immunization every14days. The tail vein blood was collected and detected the antibody levels of hemagglutinationinhibition titers of1:64after10days of secondary immune. The spleen cells from immunizedmice were fused with SP2/0myeloma cells.Indirect enzyme linked immunosorbent assay (iELISA) used to screen hybridoma cellswas developed. The best reaction conditions of iELISA were that the purified antigen wasdiluted as1:100and coated in4℃for one night, the negative and positive serum were dilutedas1:800, the temperature of the reaction was37℃and the reaction time was1.5hours. Thereaction time that enzyme sign the second antibody were diluted as1:3000was1hour. The24positive hybridoma cells were obtained by iELISA. Limiting dilution method was performedto subclone positive hybridoma cells, then9positive hybridoma cells that coμld secret McAbsstably were obtained finally after subcloning, named as A10, D1, D3, E2, E4, H1, H8, G5, G7.The stability, chromosome number, subtype, titers of the nine McAbs in the ascites andspecificity were identified. The titers of ascites of the McAbs A10, D1, H8were51200, andD3, G5as3200, and E2, H1, G7as12800, E4as25600. The specifity of the nine hybridomacells were good. The McAbs A10, D3, E2, E4, H1, H8, G5and G7were specific forhemagglutinin H1, the monoclonal antibodies D1was into positive reaction to Nerve acidenzyme N1. But all the nine McAbs were into a negative reaction with the otherhemagglutinin influenza virus and neuraminidase influenza virus and non-influenza virus. Thechromosome numbers of the nine hybridoma cells were between100and110, which wereaccording with the chromosome number of the hybridoma cells. The subtype of D1, D3, E2,E4, H8and G5were IgG1, that of A10，H1were IgG2a and G7was IgM. After detectingantigenic site, D3and G5, E4and H8correspond to the same virusantigenic sites, thesuperposition coefficient of A10and D1was90.5%, showing the good pairing， so immunochromatographic strip test for detection of H1N1subtype SIV was prepared with A10and D1.The colloidal gold particles were prepared by trisodium citric acid deoxidization and theamount of mini stability was determined by the dilution of monoclonal antibody A10. Thecombination was optimized with the conditions of the check line and the contrast linesprinkled1:2and1:20diluted protein when makes the strip. The glass fibre of AhlstromCB06and KL43were used as chromatographic materials to develop colloidal gold strip. Theresult show that the colloidal gold strip only reacted with H1N1, but did not react with theother subtype of HA and NA. The sensitivity of the immunochromatographic strip for thedetection of H1N1reached a level of0.5log2. It was higher than the hemagglutinationinhibition test, but lower than that by RT-PCR (0.25log2). Application of H1N1immunochromatographic strip for98samples collected from pigs, coincidence at81.6%withthe RT-PCR. The results show that the development of colloidal gold strip is specific andsensitive for quickly detecting H1N1subtype SIV.