The Development And Application Of Mcabs Against The NS1 Protein Of Tembusu Strain JS804 In Geese

Since April 2010, a novel contagious disease in ducks and geese, with egg drop, feed uptake decline,pull green Dilution manure and nervous system signs,caused by a newly emerged virus has spread around Anhui,Shandong,Jiangsu, Zhejiang and Fujian provinces. The virus had 100% infection,and morbidity rates and mortality rates was from 5% to 30%,laying rate is generally from 70% to 90% decline to less than 10% or even completely stop.On September 2011, outbreaking tembusu disease again in many places and the clinical symptoms similar to the diease that outbreaking in early 2010,causing breeding ducks and geese significant economic losses.In the first session of the China Institute of Veterinary waterfowl disease prevention and control seminar in 2011 will have the name of a unified disease virus duck Tembusu.After infecting the tembusu virus,mammalian secreted NS1 protein,NS1 protein can be detected in the serum before clinical symptomss does not appear. For this feature of the NS1 protein,This study was prepared geese Tembusu virus NS1 protein monoclonal antibodies and the establishment of double antibody sandwich ELIS A method for detection of the Tembusu strain JS804 in geese.At early diagnosis of infection.Thereby reducing the huge economic losses caused by the outbreak of the disease.The NS1 Protein of Tembusu strain JS804 in geese was used to inoculate BALB/c mice.After vaccinations,the splenocytes of the immunized mice were fused with murine myeloma cells SP2/0.After subcloning for 4 times, five positive hybridoma cell stains4D2,lB7,lC10,3G4,4A9.were determined by ELISA, which ELIS A titers of cell supernatants and ascites were10、10、102、102、103 �'� 103、103、104、104、105.The analysis of Western-blot and IFA indicated that the five strains of monoclonal antibody can react with the NS1 Protein of Tembusu strain JS804 in geese.Five strains of monoclonal antibody had no neutralization titers against the Tembusu strain JS804 in geese.The monoclonal antibody 4A9 have the highest ELISA value and purification effect is good. In order to establish double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA) for detection of the Tembusu strain JS804 in geese,polyclonal antibody against the Tembusu strain JS804 in geese and monoclonal antibody against the NS1 protein of the Tembusu strain JS804 in geese were used as the capture antibody and detecting antibody,respectively.The optimal dilution of the capture antibody and detecting antibody capable of detecting the Tembusu strain JS804 in geese were 1:1600 and 1:160 in the check-board titration respectively, the reaction time of sample was 1 hour,and the optimal working dilution of HRP-labelled goat-anti-mouse IgG was 1:5000. The positive standard value was 0.245(OD450nm).The coefficient of variation of reproducibility was less than 10%,and at least 386.25TCID5o/0.1mL antigen could be detectable. The ELISA had no cross-reaction with NDV、AIV、IBV、DHV and GPV. Twenty two clinical samples were detected by the ELISA and RT-PCR with agreement rate of 86.36%. The results revealed that the ELISA possessed good specificity and reproducibility,and higher sensitivity,indicating a suitable method for rapid detection of the Tembusu strain JS804 in geese.Using the double sandwich ELISA method detect serum samples.25-day-old ducks, 1、2、3、4、5、6、7、8 days after intramuscular injection the Goose Tembusu virus, collected blood per day, Using the double sandwich ELISA and RT-PCR method to detect the tembusu viruses, compared with the two methods. The test results showed that:the first day of the two methods of detection rates are negative; before the two days of positive rate is very low; The 3d、4d、5d、6d、7d、8d,Using the double sandwich ELISA to detect the virus, the positive rate are 70%、75%、50%、85%、85%、100%while the RT-PCR method were 30%、50%、30%、80%、70%、90%. The double sandwich ELISA can be detected tembusu virus earlier and more sensitive than by RT-PCR. And the overall compliance rate of the two methods is 78.75%.