Preparation Of BRSV G Protein Monoclonal Antibody And Establishment Of DAS-ELISA Method

In order to develop the DAS-ELISA method for detecting BRSV,a pair of primers was designed basing on the conservation sequence of BRSV G gene.The recombinant cloning vector and recombinant expression vector were constructed and transformed into BL21(DE3)competent cells.New Zealand white rabbits and BALB/c mice were immunized with the purified G protein as an antigen to prepare anti-G protein polyclonal and monoclonal antibodies.A DAS-ELISA method for BRSV detection was developed by using monoclonal antibodies with high specificity and double antibody sandwich with high sensitivity.The nucleotide sequence homology of the G gene was 99.83 % and the amino acid homology was 99.48 %.The target protein was the soluble protein with the size of 36 kDa,which reacted with the anti-BRSV antibody,suggesting that the expressed G protein was of the higher reactionogenicity.The highest expression level of target protein was occurred when the optimal concentration of IPTG was at 1 mM and induction at 4 h.The purification efficiency was the highest when the concentration of imidazole was 200 mM in the elution buffer.The protein concentration was 1.5 mg/mL.After rabbits were immunized with the purified protein G,the anti-G serum was harvested.The five hybridoma cell lines steadily secreting the monoclonal antibodies against G protein were obtained,namely 1E3,2B4,3F6,4B8 and 5F7,respectively.The subclass antibodies were all IgG1 with ? chain.The antibody titers in hybridoma cell culture supernatant were 1?2560,1?640,1?12800,1?320,1?640 and ascites titers were 1?256000,1?128000?1?128000,1?64000,1?128000.The square titration test determined 1E3 as a capture antibody characterized as the higher antibody titers and the best stability and polyclonal antibody as the detection antibodies.The optimal working concentration of IE3 and pAb was 2.5 ?g/mL and 5 ?g/mL,respectively.The other reaction conditions were further optimized,such as the critical value 0.239,batch,inter-assay coefficient of variation less than 10%,and the detection limit of 1.43 ?g/mL.The DAS-ELISA was used to detect the pathogens of cattle respiratory disease.The results showed that only the bovine respiratory syncytial virus had a specific response.When the DAS-ELISA and RT-PCR were used to detect 45 samples,the sensitivity,specificity of the DAS-ELISA and coincidence rate were 92.0 %,100 %,95.6 %,respectively.The results indicated that the established double antibody sandwich ELISA method for BRSV was rapid,sensitive and specific,which was suitable for the large-scale detection of clinical samples.It was the foundation of monitoring and quick diagnosis for BRSV.