Establishment Of A Double Antibodies Sandwich ELISA For Detection Of Infectious Bronchitis Virus

Infectious bronchitis virus(IBV)can infect chickens,causing infectious bronchitis(IB),which is an acute,highly contagious respiratory and genitourinary tract disease.Since it was first reported in 1931,IB has been reported all over the world,and IBV has derived a variety of serotypes.Mixed infection of IBV and other pathogens can cause serious complications,bringing huge losses to poultry industry.Antigenicity of IBV is complex,and prone to gene recombination and mutation,and cross protective efficacy between different serotypes is low,making the prevention and control of IB are very challenging.So,the development of accurate,simple and rapid diagnosis method is very important to prevent and control IB.The nucleocapsid protein,N protein,is the major structural protein of IBV,which is relatively conservative.This research focused on N protein to make anti-N polyclonal antibody from rabbit(PcAb)and anti-N monoclonal antibody from mouse(MAb),established the double antibodies sandwich ELISA method for detection of IBV,provided an effective means for the diagnosis of IBV.1.Preparation of PcAb and MAb against IBV N proteinOn the basis of IBV QXL strain,isolated and preserved by our laboratory,the sequences of the N protein were used to analysis its antigenicity.N gene fragment was amplified by the corresponding region of strong antigenicity by PCR method,then the product is connected to the cloning vector.After identification by sequencing,the recombinant expression vector was constructed with pET32a(+)vector,then was transformed into E.coli BL21(DE3),which was induced to express N protein fragment to be used as the immunogen.Adult rabbits and 6-8 weeks old BALB/C mice were immunized.When the immunized rabbits’ serum titer reached 1:104,all rabbits were executed by drawing-out all the blood in their hearts to prepare PcAb serum.When the immunized rats’ serum titer reached 1:104,its spleen was taken to be fused with SP2/0 cells.Hybridoma cells were screened by indirect ELISA,and finally obtained 4 hybridoma cell lines secreting MAb against N protein,named 6F4,7D8,3G2 and 5C9.After preparation and purification of ascites,it was further identified with Western blot,results showed that the MAb could bind with N protein specificly.2.Establishment of double antibodies sandwich ELISA method for detection of IBVThis study was based on the preparation of PcAb and MAb in the first part.With the PcAb used as coating antibody and MAb used as detecting antibody,double antibodies sandwich ELISA method was established to detect IBV.The optimized reaction conditions:the dilution of coating antibody was 1:64000,the dilution of detecting antibody was 1:4000,sealing liquid was 1%BSA-PBST,the dilution of enzyme-labeled antibody was 1:10000,chromogenic substrate reaction time was 15min.Specificity experiments showed that this method was without cross reaction with avian influenza virus(AIV),Newcastle disease virus(NDV)and avian adenovirus(FAdV).Repeatability experiments showed that intra and inter batch coefficient of variation was less than 8%.The conformance test with PCR method showed that the positive coincidence rate was 95.9%,the negative coincidence rate was 85.2%.In short,the antibodies prepared in this study could react with N protein specifically.The double antibodies sandwich ELISA method based on these antibodies was with good specificity and repeatability,and the testing results were reliable.This research provided a good diagnostic method for the prevention and control of IB.