Development Of An Indirect ELISA For NDV Antibody Detection Based On NP Protein

Newcastle disease(ND),also known as Asian chicken plague,is a highly contagious poultry disease of poultry caused by Newcastle disease virus(NDV).ND is of great economic significance due to its associated huge mortality and morbidity,and is recognized by the International OIE(OIE)as another category A poultry disease besides avian influenza.Therefore,it is particularly necessary to strengthen the diagnosis and monitoring of NDV.In addition to classical virus isolation and identification,NDV detection methods includes hemagglutination(HA),hemagglutination inhibition(HI),reverse transcription polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA).Among them,the ELISA method is currently one of the more researched and rapidly progressing technologies.Various forms of ELISA methods have been widely used in the detection of NDV antigens and antibodies.In this study,p ET-30 a was used as an expression vector to successfully prokaryotic expression and purification of NDV NP protein,and using it as a coating antigen,an indirect ELISA antibody detection method based on NDV NP protein was established,and the sensitivity and specificity of the method were further analyzed.The accuracy,reproducibility,and the coincidence rate of clinical serum samples with the HI method are designed to provide a test basis for the clinical application of the method.1.Prokaryotic expression and purification of NP protein of Newcastle disease virusUsing the Newcastle disease virus LaSota strain genome as a template,the NP gene was amplified by RT-PCR method,and the linearized expression vector p ET-30 a was digested with double enzymes.The NP target fragment and the linearized vector fragment were ligated and transformed by In-Fusion to obtain the recombinant plasmid p ET-NP.The recombinant plasmid p ET-NP,which was digested and sequenced correctly,was transformed into E.coli BL21(DE3),induced and expressed by IPTG,and a recombinant protein with a molecular weight of about 50 k D was obtained.After denaturation,the protein was purified with a His-labeled nickel chromatography column.The results of SDS-PAGE and Western Blot showed that NP protein with higher purity and better immunogenicity was obtained.2.Establishment of indirect ELISA antibody detection method for NP protein of Newcastle disease virusTo establish the indirect ELISA assay for detectimg of NDV antibodies,the purified NDV NP protein was used as the coating antigen,and the reaction conditions was optimized.The optimization results show that the coating concentration of NP protein is0.675 ?g/m L,the best dilution of serum is 1:200,the coating condition is 4 ? overnight,and the blocking solution and sample diluent are both 5% skim milk phosphate Buffer,the dilution of the tested serum is 1:200,the serum binding time is 30 min,the dilution of the enzyme-labeled secondary antibody is 1:2500,the secondary antibody binding time is 60 min,and the color development time is 15 min.This method has no cross-reaction to H9 subtype avian influenza virus(AIV),J subgroup avian leukemia virus(ALV-J),infectious bronchitis virus(IBV),avian Escherichia coli and Salmonella positive sera,and has good specificity.;Its detection sensitivity is 50 times that of the hemagglutination inhibition test(HI),and the sensitivity is good;the results of the repeatability experiment between groups show that the coefficient of variation is less than 6%,and the repeatability is good.ELISA method and HI antibody test were performed on 200 serum samples collected from farms in Hubei,Shandong,Henan and Jiangsu,and the coincidence rate was 97%.In summary,the purified NDV NP protein was obtained by prokaryotic expression purification,and was used as the coating protein.After optimizing the detection conditions,the NDV indirect ELISA antibody detection method was established.This method had high detection sensitivity,specificity and repeatability.By detecting the clinical samples,the NDV NP ELISA assay showed high agreement with that of HI assay,and could be used for the detection of NDV antibodies in clinical serum samples.