Development Of Monoclonal Antibody Agsinst Serotype 4 Avian Adenovirus And Preparation Of Gold Immunchromatography Assay Test Paper With The Monoclonal Antibody

Fowl Adenovirus serotype 4(FAdV?4)belongs to the adenoviral family,avian adenovirus,and avian adenovirus C species.The strong virulent strain of serum type 4 avian adenovirus has strong pathogenicity to chickens and cause Hydropericardium hpatits syndrome(HHS)in chickens.The mortality rate is as high as 30%to 90%.Viruses can be spread horizontally through the fecal-oral route and can also be spread vertically?It widely distributed throughout the world and has caused serious economic losses to the poultry industry in China and around the world.In this study,the prokaryotic expression system was used to express the two major structural proteins of avian adenovirus of serum type 4,the penton and the hexon,and finally two His-tagged fusion proteins were obtained.The results of SDS-PAGE showed that the size of His-penton was 78KD,and the maximum expression level was 6h after induction.The size of His-hexon was 118KD,and the maximum expression level was 3h after induction.Western-blot results showed that both of the expressed proteins were able to recognize anti-FAdV-4 positive serum and anti-His antibody with a single band.The BALB/c mice 6 to 8 weeks old were immunized subcutaneously with FAdV-4 antigen(0.5 ml each)and immunized twice at two weeks intervals.After three immunizations,the spleen cells of the mice were fused with SP2/0 myeloma cells.Using penton and hexon proteins as indirect ELISA for screening antigens,cloned and subcloned hybridoma cell lines were screened and 5 positive cell lines were screened,named 2D3,1D3,3C2,3H5 and 3D6,respectively.The ELISA titers of ascites in five positive hybridoma cell lines were:1:25600,1:12800,1:25600,1:6400,and 1:6400.Among them,4 strains(2D3,ID3,3C2,and 3H5)secreting anti-hexon protein-specific antibodies,and 1 strain(3D6)secreting antibodies specific to penton proteins.Mouse Monoclonal Antibody Isotyping identification showed that 1D3 belongs to the IgM class,2D3 and 3D6 belong to the IgG1 subclass,and 3C2 and 3H5 belong to the IgG2a subclass·The colloidal gold irnunochromatographic test strip combines the advantages of colloidal gold immunochromatographic technique,such as rapid,simple,accurate and intuitive,and the good specificity and sensitivity of monoclonal antibody technology.It is currently the most widely used diagnostic method in the field.method.In this study,anti-hexon protein 1D3 and 3C2 antibodies prepared in Chapter 2 were used as detection antibodies and labeled antibodies respectively to prepare colloidal gold immunochromatographic test strips for the detection of FAdV-4.The 1D3 antibody was labeled with colloidal gold with good homogeneity of 20 nm.The nitrocellulose membrane was coated with purified monoclonal antibody 3C2 and goat anti-mouse IgG secondary antibody.The test strips were assembled using the test line and the control line respectively.By analyzing the laboratory test results of assembled test strips,the colloidal gold immunochromatographic test strip has good sensitivity,specificity,stability and repeatability,and can be used for rapid laboratory detection of FAdV-4.It provides a reliable means and method for rapid diagnosis and immune prevention of FAdV-4 disease.