Establishment Of Colloidal Gold Detection Method For Antigen And Antibody Of Porcine Circovirus Type 2

Porcine circovirus type 2?PCV2?is the major causative agent of Postweaning multisystemic wasting syndrome?PMWS?,which has caused significant economic losses in the China's swine industry.Several detection methods for porcine circovirus have been reported,such as Electron microscopy,in situ hybridization?ISH?,immunohistochemistry?IHC?,loop-mediated isothermal amplification?LAMP?and polymerase chain reaction?PCR?.The detection methods of PCV2 antibodies mainly include indirect immunofluorescence assay?IFA?,enzyme linked immunosorbent assay?ELISA?,immunoperoxidase monolayer assay?IPMA?and gold-immunochromatography assay?GICA?.Except for GICA,the rest of the testing methods require professional personnel to carry out experimental operations and the requirements for experimental instruments are high and limited to laboratory operations.GICA not only has the outstanding advantages of easy operation and quick display of test results,but also does not require professional operation and has no restrictions on the test site.It is suitable for qualitative detection of a large number of clinical samples.In this study,the conditions for the development of PCV2 antigen colloidal gold test strips were explored.The matching scheme of test strips with good color rendering was preliminarily determined which laid a good foundation for rapid identification of PCV2.At the same time,the PCV2 antibody colloidal gold test strip was successfully developed.The test strip has good specificity,high sensitivity,high repeatability among batches and good stability.The coincidence rate with ELISA kit is high and clinically available.It provides a fast and efficient kit for the clinical diagnosis of PCV2 antibodies.1.Purification and identification of monoclonal antibody against PCV2Five kinds of monoclonal antibody-containing mouse ascites prepared?2C3,3D4,1G4,2G4,6H12?were purified.PCV2 protein was used as a sample and the purified monoclonal antibody was used as a primary antibody.Western-blot indicated that the five monoclonal antibodies were associated with PCV2 protein which laid the foundation for the preparation of the PCV2 antigen test strip.2.Development of an immunochromatographic strip for the rapid detection of antigens against PCV2Different volumes of K₂CO₃ were added to a fixed volume of colloidal gold solution to form different pH.Then the 5 different concentrations of monoclonal antibodies were sprayed on the gold standard pad as gold standard monoclonal antibody.The 5 kinds of monoclonal antibodies were coated on the T line and the goat anti-mouse IgG was coated on the C line.After being arranged and assembled,assembled into a plurality of test strips.PCV2 cytotoxicity and physiological saline were separately added for detection and color development was recorded.The test results show that after the PCV2 cytotoxicity is added,some test strips will have normal color development results,but the stability and repeatability are poor.Therefore,in the future experiments,the experimental scheme will continue to be optimized to make the antigen test strips.The establishment of the PCV2 antigen colloidal gold detection method is successfully completed.3.Prokaryotic expression and characterization of Cap proteinModification of the codon of the ORF2 gene into a codon suitable for E.coli,but the sequence of the optimized amino acid was unchanged and the optimized ORF2gene was synthesized by synthetic synthesis.Then ligated with the pET30a vector to construct a recombinant.The expression vector pET30a-ORF2 was identified by PCR,double enzyme digestion and sequencing.Finally,the vector was constructed correctly.It was named as pET30a-ORF2 prokaryotic expression vector.The recombinant plasmid pET30a-ORF2 was transformed into E.coli BL21 and the induced expression conditions of the cells were optimized.The results of SDS-PAGE showed that the target band was 37 kD,which was consistent with the expected molecular weight and achieved the target protein maximum expression.The Cap protein can be reacted with PCV2 antibody-positive serum by Western-Blot,indicating that the Cap protein expressed by the cell has immunological activity.Finally,the cells were sonicated,the supernatant was collected and purified by Ni-NTA affinity chromatography column.The collected soluble Cap protein is up to100%pure,which laid the foundation for the development of PCV2 antibody test strips.4.Development and application of an immunochromatographic strip for the rapid detection of antibodies against PCV2The purified Cap protein was labeled with colloidal gold and fixed on a gold pad.Cap protein and goat anti-mouse IgG were coated on the T and C lines,respectively.Then prepared the PCV2 antibody rapid test strip.The rapid detection method of PCV2 antibody was successfully established.PCV2 positive serum,PCV2 negative serum,PRRSV positive serum,CSFV positive serum,and PRV positive serum were used to comprehensively evaluate the specificity,sensitivity,repeatability and stability of the test strip,the comparison test with the ELISA kit was completed.The results showed that the test paper had good specificity and did not react with PRRSV,CSFV and PRV positive serum;the sensitivity and repeatability were good;the coincidence rate with ELISA kit?Rip?was as high as 95.00%.In summary,the PCV2 antibody test strip developed in this test has the advantages of simple operation and quick display of test results,It does not require professional operation and has no limitation on the use environment,it is suitable for qualitative detection of a large number of clinical samples.It further indicates that the laboratory has established a rapid detection method for mature PCV2 antibodies.