| Mink enteritis virus(MEV)and canine distemper virus(CDV)are highly infectious to mink.Mink viral enteritis and mink canine distemper caused by infection are the two most serious infectious diseases that harmful to mink farming.In the rapid diagnosis process of animal diseases,serological testing methods have the advantages of convenience,stability,high throughput.At present,there is no commercial ELISA detection kit for MEV and CDV antibodies in China,which can be used to detect MEV and CDV antibodies in mink serum.Therefore,the purpose of this study was to establish indirect ELISA detection methods for the detection of antibodies after MEV and CDV infection or vaccine immunization,respectively.In order to optimize the optimal coating antigen used in MEV antibody detection method,the MEV virus was purified by centrifugation of Ultra-15 ultrafiltration tube,purified recombinant VP2 soluble protein,and recombinant VP2 inclusion body protein as inclusion antigen of MEV indirect ELISA method,and the positive and negative sera of MEV antibody were detected.The comparative analysis confirmed that the recombinant VP2 soluble protein was identified as the best coated antigen used in MEV antibody indirect ELISA detection method,which was significantly better than that of recombinant VP2 inclusion body protein(P<0.01)and MEV virus antigen(P<0.01).The purified recombinant VP2 soluble protein was used as the coating antigen,and HRP Goat Anti-Ferret IgG(H+L)as the secondary antibody to establish an indirect ELISA detection method for MEV antibodies.The reaction conditions of the indirect ELISA method were optimized,and the specificity,sensitivity and repeatability of the MEV antibody detection were evaluated,and ELISA antibody titers were calculated by enzyme immune units(EIU).The results showed that the optimal antigen concentration of MEV indirect ELISA method was 1.425 ?g/mL,and the concentration of serum and enzyme labelled antibody was diluted 1:80 times and 1:2 000 times respectively.The established MEV indirect ELISA method can specifically detect MEV antibodies without cross-reactivity with the positive serum of mink canine distemper virus and mink Aleutian virus(ADV),and the coefficient of variation of intra batch and inter batch repeatability test is less than 5%;By comparing the parallel relationship between the MEV serum neutralizing antibody and the ELISA antibody titer,it shows tihat the MEV ELISA antibody titer<14 EIU is the antibody negative,and the MEV ELISA antibody titer?14 EIU is the MEV serum neutralizing antibody is positive.The mink distemper and mink viral enteritis live vaccine was used to immunize mink.HI test,SN test and MEV indirect ELISA method were used to detect the sera of mink in the immunized group and control group collected at different times after immunization(7 d,14 d,21 d,30 d,60 d,90 d,120 d,180 d).It was found that the MEV antibody titers of the three detection methods showed the same trend.The correlation between MEV ELISA-IgG antibody titer and neutralizing antibody titer and hemagglutination inhibition titer was analyzed.It was found that there were significant positive correlation between MEV ELISA-IgG antibody and HI antibody titer(r=0.9369,P<0.001)and SN antibody titer(r=0.9461,P<0.001).The positive correlation shows that this method can quantify the antibodies in serum and can replace the HI test and SN test methods to evaluate the immune effect of mink virus enteritis vaccine.In this study,the CDV recombinant N soluble protein was obtained from prokaryotic expression system.After purification,it was used as the inclusion antigen to establish indirect ELISA for detecting CDV antibody.The HRP Goat Anti-Ferret IgG(H+L)was used as the secondary antibody,and the reaction conditions were optimized,and the specificity,sensitivity and reproducibility were evaluated.The coincidence rate between the ELISA method and the commercialized kit was evaluated by clinical samples.The results showed that the optimal antigen concentration of the CDV indirect ELISA method was 1.383 ?g/mL,and the concentration of the serum and enzyme labelled antibody was diluted 1:80 times and 1:2 000 times respectively.The results showed that the CDV indirect ELISA method could detect the CDV antibody specifically,and had no cross reaction with the antiserum of MEV and ADV.The coefficient of variation of intra batch and inter batch repeatability tests was less than 10%,and the total coincidence rate with the commercial kit test was 94.4%. |
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