Abstract: In order to establish a NS1\| ELISA for rapid detection of antibodies against duck Tembusu virus（DTV）， a series of studies were conducted. Firstly， the non\|structural protein NS1 gene of goose Tembusu virus JS804 strain was inserted into the prokaryotic vector pET32a， then pET32a\|NS1 were transformed into Escherichia coli BL12（DE3）and the combination protein NS1 （His\|NS1） was obtained with the induction of IPTG. The fusion protein was purified by extracting the inclusion bodies with urea， and the purified protein was used as coated antigen. Based on the best working condition， the indirect ELISA method（NS1\|ELISA） to detect the antibody of duck TVD was developed. The optimized working condition included: the coating antigen of purified DTV\|NS1 protein was 1925μg·well-1， the best package condition was 37℃ for 1 h and then over night at 4℃， the best dilution of testing serum was 1∶200， the best closure time was 1 h， the dilution of HRP conjugated anti\|duck lgG was 1∶1 000，the chromogenic time was 10 min， and the cut off\|value judging negative or positive was 0346（D450）. The specific tests showed that there were no cross\|reaction to the anti\|sera against avian influenza virus， Newcastle disease virus， infectious bronchitis virus and DTV， which indicated that the NS1\|ELISA was specific to anti\|sera against DTV. The intra\|plate and inter\|plate demonstrated that the coefficient of maximum variation was 67％and 82％ respectively， which showed the method had good stability. A total of 81 samples from affected ducks were tested and 43 samples were positive detected by the NS1\|ELISA， While 48 samples showed positive detected by the E\|ELISA， which showed the method had a high sensitivity. The results revealed the NS1\|ELISA could be used for laboratory diagnosis and sera\|survey for duck Tembusu virus infection.

Key words: Tembusu virus, NS1 protein, indirect ELISA, antibody