| Avian leukosis virus subgroup K（ALV-K）is an exogenous avian leukemia virus newly emerged that it discovered in chickens of various breeds.It poses a severe threat to the breeding poultry industry in China.There is no effective vaccine or drug to treat it.Therefore,it should be specially measured and monitored.Enzyme-linked immunosorbent assay is widely available since it is efficient,convenient,and sensitive.Gp85 is the site of antigen detection for different subtypes.There have been detection methods for other subtypes of ALV.And yet,there has been no report on the double-antibody sandwich ELISA method（DAS-ELISA）for ALV-K antigen detection with monoclonal antibodies.This experiment amplified the fragment of the ALV-A/B/J/K gp85 genes to construct a recombinant plasmid.Gp85 had obtained by applying prokaryotic expression techniques.The ALV-K gp85 recombinant protein was immunized in rabbits to obtain polyclonal antibodies.This experiment immunized 6-week-old BALB/c mice by dorsal subcutaneous injection to prepare hybridoma cells,which were identified by establishing I-ELISA to prepare monoclonal antibodies.A preliminary DAS-ELISA detection method was constructed for the ALV-K gp85 and validated its sensitivity,accuracy,reproducibility,stability and practicality.The results obtained the ALV-A/B/J/K gp85 gene fragment.The recombinant plasmids ALV-B-p ET-32a-gp85 and ALV-A/J/K-p Cold-gp85 were successfully constructed and expressed by IPTG induction to gp85 recombinant proteins.The obtained proteins were 42,53,37 and 40 k Da in size and existed in the form of inclusion bodies.The potency was up to5.12×10~5with the rabbit-derived polyclonal antibodies.The rabbit-derived polyclonal antibodies specifically reacted with ALV-K and its gp85 recombinant protein.Then this study performed three rounds of screening using the limited dilution method to obtain three hybridoma cells lines.The monoclonal antibodies were named 5E12,4B10 and 3F11;the heavy chains were Ig G1,Ig G2b and Ig G2a,and the light chains were kappa.The maximum potency was up to 1.024×10~6with the monoclonal antibodies.IFA and I-ELISA results showed that 5E12 reacted with a strain of ALV-K and its gp85 recombinant protein;4B10and 3F11 reacted with a strain of ALV-A/K and its gp85 recombinant protein.In the DAS-ELISA,the polyclonal antibodies were capture antibodies and the monoclonal antibodies were detection antibodies.The testing range of antibodies was 0.977-2,000.0 ng/m L.At the gradient antigen concentration of 500,250 and 125 ng/m L,the antibody recovery of the testing method had a range of 92.1%-106.9%,86.7%-97.9%and77.4%-93.4%,respectively.This method had a high recovery rate and can accurately detect samples.Repeatability at three antigen concentration gradients of 500,250 and 125 ng/m L with intra-batch variability of 4.73%-5.71%and inter-batch variability of 3.41%-6.38%,with low batch variability and repeatable.The DAS-ELISA testing had a positive-and-negative critical value of 0.119.The coincidence rate with the commercial ALV p27 antigen detection kit was 88.37%,and the coincidence rate with the PCR detection was 90.70%.In summary,the ALV-A/B/J/K gp85 recombinant proteins had obtained in this study.The monoclonal antibody and polyclonal antibody generated by its induction reacted with ALV-K and its gp85 recombinant protein.The DAS-ELISA method had initially established.This method can be used in monitoring and controlling ALV-K,thus providing support to the epidemiological investigation. |
