Preparation Of Monoclonal Antibody Against NP Protein Of PPMV-1 And Establishment Of Blocking ELISA Method

Pigeon Newcastle disease,commonly known as pigeon plague or pigeon paramyxovirus disease,is the primary disease that harms pigeons.It is caused by the genotype VI Newcastle disease virus(NDV)and is called pigeon Newcastle disease because its symptoms are similar to chicken Newcastle disease.NDV belongs to the avian paramyxovirus type I(APMV-1)in the family Paramyxoviridae,so the gene VI NDV that causes Newcastle disease in pigeons is specifically named Pigeon paramyxovirus type I(PPMV-1).The main characteristics of pigeon Newcastle disease are nervous symptoms and digestive tract damage.The disease spreads rapidly,with high incidence rate and mortality.At present,there is a lack of commercial diagnostic kits for pigeon Newcastle disease serology,and chicken Newcastle disease detection kits are mainly used in clinical practice.Therefore,the development of specific antibody detection methods for pigeon Newcastle disease has important application value for clinical monitoring of pigeon Newcastle disease epidemic and evaluation of vaccine immune efficacy.Among the six structural proteins of PPMV-1,the nucleoprotein(NP)is the most abundant and conserved,and also has strong immunogenicity,which is theoretically ideal for the preparation of monoclonal antibodies that specifically recognize the NP protein.Therefore,in this study,NP protein was selected as the target for the preparation of monoclonal antibodies,and a blocking ELISA serological assay was established,which can achieve quantitative and rapid detection of antibody levels against Newcastle disease in pigeons.The main findings are as follows:1.Preparation of monoclonal antibodies against PPMV-1 NP proteinA virus strain PPMV-1/pigeon/Gansu/China/02/2020(referred to as GS02,Gen Bank:OM640464.1)was isolated and identified in the laboratory in the early stage.Based on this strain,the NP gene fragment was amplified and its prokaryotic expression system p ET30a-NP was constructed.After NP protein expression and purification,BALB/c mice were immunized,and spleen B cells were fused with mouse myeloma cells SP2/0.Positive cell lines were screened using ELISA,IFA,and Western blot methods.After subcloning,a total of 25 positive monoclonal cell lines were obtained,select a stable hybridoma cell line3C10E5 that secretes monoclonal antibodies and prepare ascites monoclonal antibodies by intraperitoneal injection into BALB/c mice.The monoclonal antibody 3C10E5 was identified as an Ig G1 subtype by antibody typing.The titer of the monoclonal antibody determined by ELISA can reach 10~6,and the recognition region of the monoclonal antibody is the 429-489 amino acid at the C-terminus of the NP protein.IFA and Western blot detection showed that the monoclonal antibody reacted well with the NP protein,with strong specificity,laying the foundation for the establishment of a blocking ELISA antibody detection method in the later stage.2.Establishment of PPMV-1 antibody blocking ELISA detection methodIn order to establish a blocking ELISA method,the chessboard titration method was used to determine the optimal coating amount of NP protein and the optimal dilution concentration of monoclonal antibody 3C10E5.On this basis,optimize the working conditions of blocking the dilution concentration of the serum to be tested in ELISA,blocking solution,enzyme labeled antibody dilution,and TMB color development time.The optimal reaction condition was ultimately determined to be a NP protein coating amount of50 ng/well;The dilution of monoclonal antibody 3C10E5 is 1:4000;The dilution of the serum to be tested is 1:2;Select 10%skimmed milk powder as the sealing solution;The dilution of the enzyme-linked antibody is 1:10000,and the TMB color development time is15 minutes.By testing 30 negative serum samples,the determination standard for this method is determined to be PPMV-1 antibody positive when the blocking rate of the serum to be tested is≥34.75%.Subsequently,the established methods were tested for specificity,sensitivity,and clinical samples.After testing,the method was able to accurately identify PPMV-1 positive serum,with a sensitivity of 1:64.By comparing the detection rates of clinical samples,it was determined that the detection rate of this method was consistent with the commercial reagent kit,indicating that this method has good specificity and sensitivity and can be used for clinical antibody detection of pigeon Newcastle disease.In summary,this study completed the expression and purification of PPMV-1 NP protein using a prokaryotic expression system,obtained the monoclonal antibody 3C10E5for PPMV-1 NP protein,and established a blocking ELISA method for the detection of pigeon Newcastle disease antibodies,which provides technical support for the clinical serological detection of pigeon Newcastle disease.