Establishment Of A Novel Immunochromatographic Assay Based On African Swine Fever Virus And Foot-and-mouth Disease Virus Serotype A Labeled By Immunomicrosphere

Colloidal gold as the functional carrier material combined with immunochromatographic technology and related diagnostic methods and products has the advantages of simple operation,rapid reaction,high specificity and good stability,and has been widely used in the medical inspection,food safety,veterinary drug residues,environmental monitoring and other fields.However,the immunochromatography technology based on colloidal gold particles has the deficiencies such as low sensitivity,and can be detected qualitatively.Therefore,for immunochromatography technology,it is difficult to break through the bottleneck of quantitative analysis and establish a highly sensitive,specific and rapid quantitative detection method,which can be better satisfied the needs of grass-roots production units.It has significant technical improvement value and application prospect.In this paper,immunochromatographic assays are respectively established for qualitative detection of African Swine Fever Virus(ASFV)antibody and quantitative detection of foot-and-mouth disease virus(FMDV)type A antibody.The research are as follows:1.Developing a rapid ASFV antibody test,the recombinant ASFV p30 protein with a merit of specificity and antigenicity was labeled with gold nanoparticles,and used as antigen to establish a colloidal gold immunochromatographic test strip for rapid detection of ASFV antibodies.The recombinant p30 protein and its antibodies were blotted on nitrocellulose membrane as a test line and a control line,respectively.The strip test could be accomplished with testing serum samples in 5-10 minutes.The present prepared Gold test strip showed its sensitivity as 1:256.The specificity of the test was assessed by the testing known positive serum against ASFV,and the positive serum against FMDV,CSFV,PRRSV and PCV-2 as well as the serum from a healthy pig as negative control.The results showed that the test strip had detected the ASFV positive serum specifically,while no cross reaction with the controls.Different batches were detected in triplicate,with a variance as C.V.<10%.The comparison tests of the same group of serum using a Competitive ELISA as golden standard showed that in 50 negative sera,48 samples were negative by strip test and 49 samples were negative by ELISA test,in 52ASFV antibody positive sera,50 samples were positive by strip and 51 samples were positive by ELISA.The sensitivity and specificity of the test strip were 92.52%and 90.00%,respectively.2.Develop a quantitative detection about FMDV antibody.1)Choose the most suitable diagnostic antigens.The AF/72 and Re-A/WH/09 purified 146S and VP1 expression protein,and A/GD/MM/13 VP1 expression protein were selected as the candidate antigens.Indirect ELISA and GICA were established,and 100 serum samples with clear background were tested.The results showed that the antigen activity of A/GD/MM/13 strains(P/N=1.95)was the lowest.In indirect ELISA,the sensitivity and specificity of AF/72-146S(90%,95%),and in GICA(95%,92.5%),Re-A/WH/09-146S ELISA(86.67%,87.5%)?GICA(83.33%,87.5%)?AF/72-VP1 ELISA(78.33%,75%)?GICA(90%,62.5%)?Re-A/WH/09-VP1ELISA(76.67%,70%)?GICA(85%,55%).As the most appropriate antigen,the diagnosis method by AF/72 purified 146S can obtain a satisfactory result.2)Develop a quantitative detection about FMDV-A antibody.Pt-Pd bimetal NPs are prepared by K₂Pt Cl₄and Na₂Pd Cl₄which are reduced by ascorbic acid(AA).The mixture is continuously sonicated in water bath.The purified antigen of FMDV serotype A is coupled with Pt-Pd bimetal NPs.The purified146S particles of FMDV serotype A(FMDV-146S)and the antibodies against FMDV-146S(FMDV-146S-Ab)are blotted on nitrocellulose membrane as test line and control line,respectively.The sensitivity,specificity and stability of this NPs test strip are assessed by testing known positive and negative FMDV serum and positive serum against other animals'disease.This established method could be accomplished qualitatively as well as semi-quantitative detection in 10 minutes.The sensitivity was up to 1:2⁸/test.Positive sera with known liquid-blocking ELISA(LPB-ELISA)antibody titer of 1:64,1:128,1:256 and1:512 were detected with FMDV-A test strip.After the detection,T/C of signal was scanned by immunoanalyzer.Using the antibody titer of type A positive serum samples as the abscissa and the T/C of the gold test strip as the ordinate,the standard curve was established.There was a good linear relationship between antibody titer and T/C,with correlation coefficient R²=0.9692.When the T/C value of the serum to be tested is put into the standard curve,the antibody titer of the serum can be obtained accordingly.The comparison tests of the same group of serum using gold test strip showed that in 50 FMDV-A sera,49 samples were positive by Pt-Pd NPs test strip and 48 samples were positive by gold test strip,in 22 FMDV antibody negative sera,22 samples were negative by two strips,in 36 FMDV-O?Asia?sera,34 samples were positive by Pt-Pd NPs test strip and 31 samples were positive by gold test strip,The sensitivity and specificity of Pt-Pd NPs test strip were 98%and 94.8%,respectively.The difference between batches are small,with a variance as C.V.<15%.Parallel tests of the positive FMDV serum by LPB-ELISA were performed.Test serum samples show that the strip results are coincident with LPB-ELISA,R²=0.9112.