The Preparation Of Canine Distemper Virus Monoclonal Antibody

Canine distemper(CD) is an highly contagious disease,which is caused by canine distemper virus(CDV). The characteristics of the disease have strong infectivity, higher pathogenicity and various clinical symptom, which are easily infected with the bacterias and viruses, and the mortality reach up to 80%. At present, there is no method which is rapid, sensitive and easy diagnostic in the market.It was said that supernal connectivity between the high-performance immunoglobulin and the antigen.So we could make use of hybridoma technique,and make into the Canine distemper virus monoclonal antibody.In spite of there were many methods detected in amynology by antiserum , the antiserum could not provid orthonormal reagent,so it is hard to be make use of the large industrialization and the productions. Monoclonal antibody come form the same cell,so it could. Monoclonal antibody utend the same antigenic determinant and no only antibody.so that the detection had higer specificness.The antibody titer of ascites were attainable to the antibody titer of the blood serum hundreds and thousands fold and it easy made into.Once prepareing the hybirdoma cells,it could be preserved in the liquid nitrogen long times,so it were incessantly supply the standard agent for the detection and production.The test has prepared monoclonal antibody of canine distemper virus, which has highly neutral titer. It will be combined with immune colloidal gold technique in the future, wich will establish colloidal gold diagnostic test paper of canine distemper. It will provide a rapid, sensitive and easy diagnostic method in the clinic, at the same time, it also will provide basis and guidance for clinical treatment.The growth properties and pathogenic characteristic of canine distemper were cytopathogence,including cellular necrosis,syncytium formation and inliusion bodieswithin infected cultures.Cytoplasmic inclusion bodies were detected 4 days positinoculation,which was followed the intranuclear inclusion body formation.The optimal conditions of canine distemper on Vero cells was at 37℃for 5～6days. By electron microscopy, the process of virus morphogenesis was consistent with literature, there are acervate nucleocapsid, the germination and multiplication of plasmalemma were found, electron pyknotics were found in the later period. Cytopathy was very contiguous with literature about CPV in Vero cell.Spleen cells form a BALB/C mouse immunied with CDV were fused with SP2/0 myeeloma cells by polyethylene glycol,After screening and cloing, 3 hybrid cell lines steadily secreting monoclonal antibodies were obtiained. they were 1F3,2A6,2F7.By the identification ,the Ig classes of McAbs were IgG1(1F3,2A6) ,IgG2b(2F7).The neutralizing antibody tites of 3 ascites range were 1:3236,1:1600 and 1:1600. All of McAbs were good in specificity and no cross-reactions with CPV were found.The harvest monoclonal antibodies were purified by a saturated ammonium sulfate percipition,the recovery rate and activity were analyzed by BAC and the sandwich-ELISA. The average recovery of purified monoclonal antibodies was31.2%.The valency was no difference with the purified and unpurified monoclonal antibodies, The valency of the purified monoclonal antibodies was not degrade.