Development Of Monoclonal Antibodies Against Bovine IFN-γ And Establishment Of Antigen Capture ELISA For Bovine IFN-γ Detection

Interferon (IFN) is a kind of inducing glycoprotein which possesses high bioactivity and broad-spectrum antiviral activity on the homogeneity animal cells, its function is controlled and regulated by cell genome. IFN-γtest is established on the base of IFN-γMcAb which can detect the qualitation and quantity of interferon gamma. IFN-γand its monoclonal antibody also have a broad application in veterinary medicine. Some foreign scientists have reported that monoclonal antibody has been used for the research of bovine diseases. Some researches have been reported in China in recent years, but a few researches for detection. This study cloned and expressed recombinant BoIFN-γ(bovine interferon gamma), produced monoclonal antibodies against BoIFN-γand established a sandwich direct ELISA to detect BoIFN-γ.Our study includes two parts:Part one: The BoIFN-γgene fragment was cloned into prokaryotic expression plasmid. The recombinant protein was purified by affinity chromatography and used to immunize BALB/c mice. After three to five sub-clone,two hybridomas 2G6 and 5F9 secreting monoclonal antibodies to BoIFN-γwere established by ELISA . The two McAbs recognized BoIFN-γspecifically by western blot and indirect ELISA. The antiviral activity of BoIFN-γexpressed by recombinant baculovirus could be blocked by the two McAb ascites.The additivity ELISA demonstrated that the two McAbs recognized different antigenic regions.Part two: A sandwich ELISA for detection of BoIFN-γwas developed utilizing 2G6 McAb and HRP-labled 5F9 McAb which could detect BoIFN-γspecifically.The optimal working condition was listed as below. The dilution of capture McAb was 0.127μg/well, the standard protein was diluted to 1:20 and the dilution of HRP-labeled 5F9 was 0.130μg/well , the reaction time for coating antibody and HRP-labled 5F9 were all 1.0h in the assay,the coating buffer was 20mM Tris-cl pH8.5,the blocking solution was 3% gelatin from cold water fish skin, CV of intra-plate and inter-plate was lower than 10%. All the specific test in this paper demonstrated the two McAbs had good specificity .The result showed that the ELISA had a detection limit of 98.43ng/mL BoIFN-γexpressed by pET-30a(+)-BoIFN-γin BL21, 2IU/100μL BoIFN-γexpressed by rBac-BoIFN-γ, 10IU/100μL BoIFN-γexpressed by r-NDV-lasota-BoIFN-γ.After accelerated temperature studies for protein activity of 2G6 coating microplate the result showed that it could endure 30℃for 4 months.