Identification And Monoclonal Antibody Preparation Of Canine Distemper Virus

Canine distemper (CD) is a highly contagious and fatal disease of dogs or other susceptible animals which infected by canine distemper virus (CDV). At present, canine distemper is pandemic in China and becomes one of the most serious diseases for dog breeding industry. According to the characteristics of cell culture,the study isolated a strain of canine distemper virus (CDV) attenuated strain from Second United attenuated sample in Vero cells using hereroviusantiserum blocking assaying. The CPE in Vero cell featuring gathered shedding, rounding, net-balloon and death appeared steadily after generation cultivation.The diseased cells identified by RT-PCR showed positive.The physics and chemistry experiments showed that, the virus was inactivated 56℃for 30min, more sensitive to changes of acidity. The strain held its virμLence stability in pH7.0～8.0 environment but sensitive above pH10.0 and below pH4.0. CDV is an enveloped virus,lossed its infection activity after 20 minutes with chloroform.The HA test show that,CDV could not agglutinate red blood cell of chicken, pig, sheep and rabbit. The TCID50 of CDV attenuated strain is 10-625/mL.The 6 to 8 weeks old BALB/c female mice were immunized subcutaneously with CDV Oonderstepoort strain, which was purified and concentrated with polyethylene glycol. After being immunized three times, the mice were boosted intraveinally with equal the dose for the final immunization. Three days later, spleen cells from immunized mice were fused with SP2/0 myeloma cells. After repeated screening and subcloning,5 hybridoma cell lines were obtained which coμLd steadily secrete monoclonal antibody against CDV. They were named:1G5,2D7,3B6,4C7 and 4G5。Monoclonal antibodies in 5 lines all had higher specificity to CDV, which did not react with adenovirus typeⅡand canine parvovirus. The IFA test showed that all the 5 mABs can react with Vero cells infected by CDV. The ascites titers of 5 hybridoma cell lines were 1G5>100x210,2D7>100×29,3B6>100×274C7>100×28,4G5>100x210, the titer of cell cμLture supernatant was 28～29. The heavy chains in monoclonal antibodies of 5myeloma cell lines belonged to IgG1 and light chains belonged toκchains.