Preparation And Identification Of Monoclonal Antibodies Against M Protein Of Infectious Bronchitis Viruse The Strain Sczy3

Avian infectious bronchitis (IB) caused by the infectious bronchitis virus (IBV) is a highly acute and cotact disease,which is worldwide distribution. It can cause coughing, sneezing, tracheal rales, decrease egg production and quality,and decline in the weight gain and feed conversion of chickens.It have a serious harm to the world poultry industry. IBV is a typical coronavirus, Because IBV exists as dozens of serotypes, the antigenic of IBV is complex, the frequent point mutations and genomes recombination, currently, many new serotypes and genotypes of IBV have been detected. It is hard to diagnosis, prevent and control IB. Monoclonal antibody (McAb) have high stability, homogeneity, and strong specificity, so it is better than the conventional serum to study the biological characteristics of IBV and the detection of IBV. In recent years, LX4genotype IBV is popular in China and European countries. The strain Sczy3was isolated in Ya’an area from Sichuan in2009, and it is a LX4genotype on basis of the sequence comparison of S1gene. Membrane (M) protein is an important structural protein of IBV. In this study, we prepared McAbs against the M protein of IBV Sczy3strain, and it has an important significance to improve the IBV diagnostic level and epitope studies.In this study, IBV Sczy3strain was inoculated into the9to11days of non-immune chicken embryos for virus multiplication culture and was purifed by differential centrifugation. The6to8weeks male BALB/c mice were immunized with purified IBV Sczy3strain, and the spleen cells of immunized mouse were fused with myeloma cells SP/20after the ELISA antibody titers of the mouses serums were greater than104. Indirect ELISA method was used for the selection of wells secreting McAb, and positive wells were further subcloned4times by limiting dilution method. Two McAbs against IBV were obtained and named as2C10and7H6. McAbs were prepared by ascites induction method.2010was purified by caprylic acid and ammonium sulfate precipitation,7H6was purified by caprylic acid-ammonium cold ethanol precipitation, and the purification effect is good. The titers of two McAbs cell culture supernatants and purified ascites were determined by indirect ELISA method. The ELISA antibody titers of2C10cell culture supernatant was greater than1:5120and the ELISA antibody titers of its ascite was greater than106. The ELISA antibody titers of7H6cell culture supernatant was greater than1:2560and the ELISA antibody titers of its ascite was greater than105.the isotype of2C10and7H6were IgM and IgG1,respectively, The two McAbs had good specificity. Western-blot assay showed that this two McAbs could reacted specifically with M protein of IBV. Specificity test showed that this two McAbs could react with IBV of five genotypes, but not with Newcastle disease virus (NDV), Avian influenza virus subtype H9(H9AIV), Avian influenza virus subtype H5(H5AIV) antigen and Avian infectious bursal disease virus (IBDV).Two McAbs against IBV M protein were obtained successfully and they could be used for research M protein and diagnosis of IBV. This two McAbs could be used for the establishment of antigen-capture ELISA of IBV.