Establishment And Application Of Chemiluminescence Detection Method For Antibody Against Classical Swine Fever Virus

Classical Swine Fever(CSF)is one of the important pig diseases caused by Classical Swine Fever Virus(CSFV),which brings huge economic losses to the swine industry.In recent years,the current situation of virulent breeding pigs,congenital infection of piglets,emergence of mild CSF,increase of atypical CSF,variation of epidemic strains and the reduction of vaccine effectiveness caused by the introduction of other genotypes of CSF have brought great challenges to the eradication and purification of CSF in China.Chemiluminescence immunoassay(CLLA)is a new technique of Chemiluminescence combined with immunoreactivity.It has many advantages,such as a wide linear range,no radioactive contamination,high sensitivity,strong specificity,rapid and simple,and easy to operate.Therefore,in this study,the E2 protein of CSFC was used as antigen to establish a CLIA antibody detection method with high sensitive and specificity properties,which provide a new technical method for the eradication and purification of CSF.1.Acquisition of truncated E2 recombinant protein.The extracellular region of E2 gene was amplified by RT-PCR,and the recombinant eukaryotic expression plasmid pc DNA3.1-His-E2 was constructed and then was transfected into CHO cells to obtain secretory E2 protein.The E2 protein with His label was purified by Ni column,and the truncated E2 recombinant protein was obtained.Western-blot results showed that the E2 protein had good reactivity with CSFV positive serum.2.Establishment of CSFV CLIA antibody detection method.The obtained E2 protein,goat anti-pig Ig G-HRP and Luminol solution were used as the coating antigen,HRP-conjugated antibody and the luminescent substrate,respectively.By optimizing the dilution solution,blocking solution,antigen coating concentration,serum dilution ratio,reaction conditions and the optimal working concentration of HRP-conjugated antibody,a method for detecting CSFV CLIA antibody was established.This method can complete the detection at room temperature for 20 min.There was no cross reaction with positive sera of Foot-and-Mouth Disease type A(FMDV A),type O virus(FMDV O),Porcine Reproductive and Respiratory Syndrome Virus(PRRSV),Seneca Virus(SVA),African Swine Fever Virus(ASFV)and Bovine Viral Diarrhea Virus(BVDV),with high specificity.The intra-batch coefficient of variation was 1.80% ? 6.88%,the inter-batch coefficient of variation was 1.11% ? 9.18%,and the coefficient of variation was less than 10%,indicating its possesses good reproducibility.The sensitivity was1:32,which was comparable to the sensitivity of commercial CSFV antibody detection kits.3.Through the detection of 152 field pig serum samples and comparison with the commercial CSFV antibody detection kit,the Kappa value was 0.929 with a high degree of consistency.In conclusion,the CSFV CLIA antibody detection method established in this study has strong specificity,high sensitivity,good reproducibility,simple and rapid,and can be applied to the detection of CSFV antibodies.