Development Of Rapid Test Strip For Detecting Classical Swine Fever Virus Antigen Antibodies

Classical swine fever（CSF）is a highly contagious and fatal infectious diseases,which is caused by classical swine fever virus（CSFV）.The prevention and control measures of CSFV are based on rabbit attenuated classical swine fever vaccine immunization in China.The vaccine is safe and valid.The vaccine can not only produces complete cross-immune protection against different genotype CSFV strain,but also can reduce or inhibit maternal antibody interference by appropriately increasing the immune dose.Monitoring the level of antibody,which designs the immunization procedures individual and improves the level of herd immunity,has become the key technology for the prevention and control of classical swine fever and the evaluation of vaccine immunity.In addition,the epidemic of atypical swine fever is more common currently,and the phenomenon of mixed infection with other swine diseases is becoming increasingly serious.Diagnosing CSF must rely on laboratory diagnostic methods,for which it is difficult to diagnose CSF by only relying on clinical symptoms and pathological anatomy.This study focuses on the development of immunochromatography test strips used for rapid antibody detection and early diagnosis of CSFV.The main contents in this study are as follows:1.Preparation and immunogenicity analysis of the E2 protein of classical swine fever virusUsing the Bac-to-Bac baculovirus expression system,CSFV E2 protein deleting the cross-membrane region was expressed and secreted by gp67 signal peptide.With a high glycosylation level,it was closer to the native conformation of E2 protein.CSFV E2 protein had a high yield of 25 mg/L.The level of induced immune protection was assessed by rabbit immunization assay.The rabbits were immuned with CSFV E2 protein to produce high levels of E2-specific and neutralizing antibodies.Therefore,the E2 protein prepared in this study had a good immunogenicity.It had laid a good foundation for the subsequent detection and study of CSFV antibody and the preparation of monoclonal antibody.2.Development of classical swine fever virus antibody test stripAn immunochromatographic test strip for rapid detection of antibodies to CSFV was developed by using CSFV E2 protein that was prepared through a baculovirus expression system.By optimization of the CSFV antibody test strip,the accuracy,sensitivity and stability of it can meet the needs of commercialization.After the dilution of CSFV standard positive serum,the limit of detection（LOD）of the CSFV antibody test strip was 1:102400.CSFV antibody test strip showed good specificity and did not cross-react with positive serum of other related viruses.The CSFV antibody test strip had good reproducibility and reliability,and the CV of the intra-group and inter-group was less than 15%.It was kept at room temperature for 15 months,and the characteristics,sensitivity,specificity,and uniformity of the CSFV antibody test strip were unchanged.813 pig serum samples were detected by CSFV antibody test strip and IDEXX CSFV Ab Test Kit.The specificity of the CSFV antibody test strip was 91.04%（61/67）,the sensitivity 97.72%（729/746）and the coincidence rate 97.17%（790/813）.Therefore,the CSFV antibody test strip developed in this study is convenient,fast,accurate,high sensitivity and high specificity,and can be used for clinical CSF antibody detection and provide a technical support for CSF prevention and control.3.Preparation and characterization of monoclonal antibody to CSFV E2 proteinBALB/c mice were immunized by using 10μg CSFV E2 protein that was expressed in the baculovirus expression system.Splenocytes of mouse postimmunized were fused with myeloma cells Sp2/0 by conventional methods.Hybridoma cells were screened by indirect immunofluorescence.The positive hybridom cells were subcloned by limiting dilution assay for three times to obtain two monoclonal antibody hybridoma cell lines,which could stably secrete antibodies against CSFV E2.The two were named 12B5 and 19A3,which specifically identified the CSFV Shimen strain.Monoclonal antibodies were generated in enterocoelia of mice,and ascites was purified to yield high concentration and high purity m Abs.After subtype identification,heavy chains of the two m Ab were Ig G1,and light chains were Kappa.By indirect ELISA,the m Ab titer was 1:102400,while by IFA,the12B5 titer was 1:6400,and the 19A3 titer was 1:1600.33 overlapping peptides was designed by CSFV E2 protein（1-331aa）and 8 hydrophilic region peptides was predicted by bioinformatics B cell epitope prediction software.Epitopes of m Ab 12B5 was identifed with the above peptides by overlapping peptide method.The results of indirect ELISA and dot-blot indicated that the epitope recognized by m Ab 12B5 was¹⁴⁷LRTEVVKTFRREKPFPHRMDC¹⁶⁷.The epitope wasβ-sheet and loop in the E2 protein structure,which had hydrophilicity and antigenicity.4.Development of classical swine fever virus test stripAn immunochromatographic test strip for rapid detection of CSFV antigen was developed by using high sensitivity and high affinity monoclonal antibody 12B5.After gradient-diluted CSFV Shimen cell cultures were tested,the LOD of CSFV antigen test strips was 1:128 and a viral load was 1.108×10³copies/μL.The test strip showed good specificity and did not cross-react with cell cultures of other major associated viruses.According to the results of CSFV real-time RT-PCR,50 clinical samples were tested by using the CSFV antigen test strip and CSFV real-time RT-PCR.The specificity of CSFV antigen test strip was 100%（35/35）,the sensitivity 86.67%（12/15）and the coincidence rate94%（47/50）.Therefore,the CSFV test strip is convenient,fast,high sensitivity and high specificity,which is suitable for clinical preliminary detection of CSFV antigen.In conclusion,this study developed an immunochromatographic test strip for rapid CSFV antibody detection,which could be widely used for clinical CSFV antibody detection and had high accuracy,sensitivity and stability.It also developed an immunochromatography test strip for rapid detection of CSFV antigen by using specific monoclonal antibody and labeled m Ab-polyclonal antibody intercept detection mode.High sensitivity and specificity were showed in the detection of clinical tissue samples.CSFV real-time RT-PCR detection method can be replaced by CSFV test strip for the differential diagnosis of clinical CSFV and other related diseases.