Development Of An Immunochromatographic Strip For Rapid Detecting Antibodies Against ALV-J

Subgroup J Avian Leukosis disease is a neoplastic infectious disease caused by subgroup J Avian Leukosis Virus(ALV-J).In 1988,ALV-J was firstly isolatedand identified in United Kingdom,it spread rapidly to other countries.ALV-J could spread horizontally and vertically.The main clinical manifestations in ALV-J with a variety of neoplastic lesions was been showed,which will lead to the slow growing,reduced egg production and immunosuppression.It will bring huge economic losses to the Poultry industry.Since the isolation and identification of ALV-J in China in 1999,ALV-J was prevalent in chicken group,at the same time it spread to the local chicken flocks.So avian leucosis disease became one of the most serious infectious diseases in China,The development of a specifial,rapid and sensitive detection technology is an effective measure to prevent and control ALV-J.At present,P27 detection ELISA kits are commonly used in the eradication of ALV.The methods about detection of ALV-J's antibodies were non-specific and unstable.Therefore,developing an specific,rapid and sensitive method to detect ALV-J's antibodies is necessary.1.Preparation of mouse anti-chicken IgG monoclonal antibodyChicken IgG was isolated by saturated ammonium sulfate precipitation method and Sephadex gel purification method.Then,the purity of chicken IgG was identified by SDS-PAGE.Seven-week old BALB/c mice were immunized with chicken IgG every 15 days for three times.Then the spleen cells were collected and fused with SP2/0 cells.Four hybridoma cells which capable of stably secreting anti-chicken IgG antibodies were screened by ELISA and subcloned three times.These four hybridoma cells were named as CIgG-1H7,CIgG-2C9,CIgG-3G3,and CIgG-5B3 respectivly.The result indicated that the CIgG-1H7,CIgG-3G3 and CIgG-5B3 were IgG1,and CIgG-2C9 was IgG2a.The light chain subclasses of the four monoclonal antibodies were all ?-type.The titer of ascites fluid was 1:80000?1:320000 by indirect ELISA.All four monoclonal antibodies recognized with the heavy chain of chicken IgG in Western blot.MAbs CIgG-1H7 and CIgG-2C9 shows no cross reactivity with other animal IgG in indirect ELISA.MAbs CIgG-3G3 and CIgG-5B3 showed specific fluorescence in indirect immunofluorescence assay.The monoclonal antibodies prepared in this study provide reliable materials for the development of diagnostic reagents for chicken diseases.2.Development of a test strip for rapid detection of ALV-J antibodyThis study was based on the identification of the specific antigenic epitopes of the ALV-J envelope protein gp85,a synthetic peptide was used to establish a ELISA kit and to detect the ALV-J antibody in serum.The peptide ALV-J-P2 was printed on the test line.The CIgG-5B3 monoclonal antibody prepared in the content 1 was selected as the gold-conjugated antibody.The antibody conjugated complex was prepared and sprayed on the gold-marked pad of the test strip.Goat anti-mouse IgG printed on the NC membrane as the control line of the test strip.Finally,the test strip was prepared by spraying,assembling and cutting,which was able to detect the specific antibody of ALV-J in chicken serum quickly and sensitively.The test strip could detect antibodies in serum from ALV-J JS09GY07 infected chicken.