| Newcastle disease (Newcastle Disease, ND) and infectious bronchitis (Infectious BronchitiS, IB) are the highly contagious respiratory diseases for the poultry. Both of them not only affect some kinds of poultry, but also can infect wild bird. They have brought huge economic losses to the global poultry industry. Therefore, we have been insisiting on preventing this type of disease for a few years.Newcastle disease virus (NDV) is the pathogen of Newcastle disease, which encode six structural protein, the Hemagglutinin neuraminidase glycoprotein (HN) is one of the most improtant immunogen; Avian infectious bronchitis virus can encode four structural proteins, Nucleocaps protein(N) as the IBV internal protein is more conservative, which has good immunogenicity. For this reason, in this study NDV-HN and IBV-N are selected as immunogens to prepare monoclonal antibody used to the colloidal gold immunochromatography technology. A method will be established which used to rapid differential diagnosis between NDV and IBV. The results were detailed as following:1. The preparation and purification of monoclonal antibodiesthe recombinant plasmid of NDV-HN gene which lacked the signal peptide and transmembrane region (The target fragment of about1590bp) was used to expressed the protein at the size of80KDa. The recombinant plasmid (The target fragment of about1230bp) of IBV-N expressed the protein at the size of80KDa. Purified the protein was used to immunize BALB/c mice, respectively. NDV-HN highly-specific monoclonal antibody was obtained by hybridoma technique, named2F2B2、4A10F11、4E2A9、4F4E2, and the IBV-N-McAb was named2E2A2. All of the ELIS A titer was higher than1:16000, the HI titer of2F2B2、4A10F11、4E2A9and4F4E2were above1:1024, but2E2A2was only1:128.2F2B2、4A10F11、4E2A9、4F4E2was high-specific, but2E2A2have faint corss-reaction with AIV-H9.2. The preparation and purification of polyclonal antibodiesIt was preserved that the kidney-type IBV was separated from chicken (XiaoGan HuBei) in Laboratory. Throught chicken embryos passage, proliferated allantoic fluid was effected by1%trypsin in37℃for1h, and the HA titer was213, purified allantoic fluid was emulsified with Frund’s adjuvant to immunize chicken, The antiserum againsted IBV was dissociated and purified by (NH4)2SO4precipitation and G-200repectively, which was highly-specific proved by indirect ELISA, and The HI titer was above1:8000. 3. establishment of immunochromatographic strips for rapid detectionThe colloidal gold particles were made by sodium citrate reduction method, the maximum adsorption peaks was at523.2nm. In the preparation of colloidal gold immunoassay, it chose monoclonal antibody to label. Determining of optimum pH was that colloidal gold per ml added eight micrograms of potassium carbonate (0.1M). The optimal protein dosage was7.2μg/ml. The0.01mol/LpH7.2PB+3%Methanol was used to be coating liquid, they were marked in the NC membrane of NDV monoclonal antibody as T1line, IBV polyclone antibody as T2line. The choice of blocking solution was0.1MpH7.4PBS+2.5%Sucose+2%BSA+0.02%NaN3+0.75%PVPK30+0.5%PEG-2000. The test strip had been assembled, the testing result show it had high specificity, sensitivity and stability.4. animal experimentAnimal testing was divided into three test groups and a control group. Each group was consisting of10chicken, two of the test groups immuned single virus separately, the third immuned mixture virus of NDV and IBV, and the control group immuned physiological saline, through mucosal barrier into body (0.2ml/bird). After inoculation, throat swab and cloacal swab were collected every day. NDV can be detected at third day by the detection strip and IBV can be detected at fifth day by the detection strip. After slaughter, it was found chicken had varying degrees of pathological symptoms, such as bleeding, swelling. Detecting all groups of throat swab and cloacal swab by the test strip, the positive ratio is93.3%with a coincidence at93.3%with indirect ELISA. |
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