Establishment And Application Of Latex Nanoparticle-enhanced Turbidimetric Immunoassay For Quantitative Detection Of Classical Swine Fever Virus E2 Protein

Classical swine fever(CSF)is popular worldwide.The main pathological features of the diseased pigs are extensive bleeding,infarction and necrosis of the internal organs,and the mortality rate is over 90%,seriously endangering the pig industry.At present,the control of CSF is still based on vaccination.Classical swine fever virus(CSFV)E2 protein is the main protective antigen of CSFV and has become a research hotspot for the development of new vaccines.Protein content detection is the key to the quality control of the CSFV E2 protein subunit vaccine semi-finished product.However,the conventional protein quantification method is aimed at detecting the total protein in the solution and cannot accurately quantify the target protein.In this study,in order to solve this problem,Latex nanoparticle-enhanced turbidimetric immunoassay(LTIA)against CSFV E2 protein was established by using monoclonal antibodies against different epitopes of CSFV E2 protein.The method is capable of specifically detecting a protein of interest in a sample,and is simple and rapid.In this study,three hybridoma cell lines of CSFV E2 protein monoclonal antibodies(15A9,14F12 and 12H12)screened in the laboratory were firstly resuscitated to prepare ascites,and their titers were determined by indirect immunofluorescence(IFA).Ascites was purified by G protein affinity chromatography,and its purity and concentration were identified by polyacrylamide gel electrophoresis(SDS-PAGE)and bisquinolinecarboxylic acid(BCA).Initially,the monoclonal antibody pairing screening was carried out by double antibody sandwich ELISA,and then using latex enhanced immunoturbidimetry to further screen monoclonal antibody pairing to determine the best pair of monoclonal antibodies for subsequent experiments.In order to further improve the sensitivity and accuracy of the detection method,the established latex-enhanced immunoturbidimetric method was used to optimize the reaction conditions and system.The latex-enhanced immunoturbidimetric assay kit was assembled according to the optimal conditions obtained above,and the linear range and standard curve of the kit were determined,and the minimum detection limit,accuracy and precision were verified.Finally,CSFV E2 protein samples expressed by CHO cell system were collected and detected by SDS-PAGE,BCA method and LTIA method established in this experiment,and methodological comparison was carried out.The results of the study showed that the linearity of the standard curve of the method was better when the CSFV E2 monoclonal antibody 12H12 and 15A9 were used to label the latex microspheres,the latex particle size was 150 nm,and the monoclonal antibody labeling ratio was 10:1.The reaction system of this method was 150 ?L of sample diluent(R1),5 ?L of CSFV E2 protein standard and 50 ?L of sensitized latex particles(R2),and a reaction time of 200 s.In addition,the linear range of the kit was 25 ?g/mL~0.5 mg/mL,the recovery range was 91%~100%,the intra-assay coefficient of variation was under 15%,and the inter-assay coefficient of variation was under 10%.The detection results of the unpurified CSFV E2 protein sample were consistent with the results of the BCA method and the SDS-PAGE method,and the detection requirements of the sample were satisfied.