Preparation Of Monoclonal Antibodies Against Mink Enteritis Virus And Establishment Of Double-antibody Sandwich ELISA

Mink enteritis virus(MEV) was the pathogen of mink viral enteritis.MEV is a member of the family parvoviridae,genus parvovirus.Under natural conditions,different species and different ages of mink both are susceptible,principally affecting kits.Mink viral enteritis caused by MEV was reported in all the raise mink country.With the development of fur animal farming industry, viral enteritis has been one of the three serious diseases of mink,and has been brought huge economic losses to mink breeding industry.In this experiment,I took the mink enteritis virus strain SMPV-11,which was isolated and preserved by our laboratory,immunized healthy mink,and collected intestinal contents at postinfection day 5.After separation and purification,virus particle of diameters 20～24 nm was detected through negative staining by electron microscope,and the highest titer was 2¹³ by hemagglutination test.BALB/c mice were immunized with the purified mink enteritis virus antigen,and when the mouse serum titer was 1:2.56×10⁵ by indirect enzyme linked immunosorbent assay(ELISA),Spleen cells were taken to fuse with SP2/0 myeloma cells and hybridoma cells were cloned 3～4 times by using limited dilution method.As a result,8 hybridoma cell lines steadily secreting monoclonal antibodies against SMPV-11 strain were obtained, designated as D3,H5,F5,F4,A9,B7,H11 and H8 respectively.The ELISA titers of all the eight strains monocloal antibodies were between 1:6.4×10³ and 1:2.56×10⁴.The specificity test proved that all the monocloal antibodies were specific to mink enteritis virus(MEV),canine parvoviurs(CPV)and feline panleukopenia virus(FPV),whereas they did not react with canine distemper virus(CDV),aleutian disease virus(ADV)and canine adenovirus(CAV).All the monocloal antibodies were identified to be IgG1 and IgG2a by using Mouse Mab Isotyping test kit.The number of chromosomes of hybridoma cell was about 105.The indirect immunofluorescence assay proved that all the eight monocloal antibodies induced bright green fluorescence in F81.There were specific reaction zone on 63.5 ku except for D3,H5 and H11 by western-blotting anailsis.Neutralization test proved that neutralization titers of F5 and A9 were 1:27 and 1:23,and the other six strains monoclonal antibodies had no neutralizing capacity.And the HI titer was 2¹¹ by hemagglutination inhibition test.The anti-MEV monoclonal antibody laid the foundation for further study of MEV and establishment of quick diagnostic method for MEV.A double-antibody sandwich enzyme-linked immunoabsorbent assay(DAS-ELISA) was established for measuring MEV by using monoclonal antibody F5 and mink anti-MEV hyperimmune serum.In this double-antibody sandwich ELISA method,the optimal concentration of capture McAb(F5) was about 241.7μg/mL(diluted ratio was 1:20),the optimal concentration of mink anti-MEV hyperimmune serum was 4.676μg/mL(diluted ratio was 1:1 600),The minimal MEV antigen which could be detected by this method was 2.5μg/mL.No cross-reaction was observed with CDV,ADV and CAV,and high coincidence rate was shown by reaction with known positive and negtive MEV antigens.The assay can be used for detecting the active antigen during vaccine-purifying process and it has characteristics of fast to get result,easy to handle,and high sensitivity and specificity.