Preparation And Application Of The Monoclonal Antibodies Against Canine Distemper Virus

Canine distemper(CD), which is caused by canine distemper virus(CDV), is an highly contagious and fatal disease. The characteristics of the disease are associated with strong infectivity, high morbidity and mortality. CDV can spread across large different species and cause various clinical symptom. Furthermore, infection may lead to systemic disease with severe immunosuppression. CD is one of the most severe infectious diseases in canine farming, far cultivation and wildlife conservation. So it is important to develop rapid and specific detection methods for the prevention and treatment of CD. In this study, the monoclonal antibodies against canine distemper virus were preparated. Based on the monoclonal antibodies, sandwich ELIS A and colloidal gold immunochromatographic assay for detecting canine distemper virus were established. The paper contains five parts:Part one:Isolation of canine distemper virus and analysis of the nucleocapsid protein geneCanine distemper virus(CDV) was isolated from the lung and liver samples of an atypical clinical case of canine distemper(CD). The nucleocapsid protein (N) gene of the CDV was amplified by RT-PCR with a pair of primers designed based on the N gene sequence of CDV reference strains in GenBank. The sequence analysis demonstrated that the homology of the CDV N gene with other12virulent reference strains in GenBank were from96.6%to99.2%in nucleotide and97.9%to99.4%in amino acid sequence, and shared93.2%to93.6%nucleotide sequence homology and96.4%to97.5%amino acid sequence with that of4attenuated vaccine stains. The phylogenetic tree based on the sequences of the N gene showed that the CDV was in the same subgroup with the virulent strains. Furthermore, the N gene from the CDV was sub-cloned into pET-28a(+) and expressed in E. coli. SDS-PAGE analysis indicated that the recombinant N protein was62kDa and mainly existed as inclusion bodies in E. coli. Western blotting showed that the recombinant N protein could be recognized by CDV positive serum.Part two:Establishment of the hybridomas secreting monoclonal antibodies against canine distemper virusThe splenocyte of BALB/c mice immunized with purified canine distemper virus (CDV) were fused with SP2/0cells. Five hybridomas secreting monoclonal antibodies (mAb) against CDV, designated as3H11,1D7,2C9,1F8and3G3, were selected with inderect ELISA and cloned by the method of limiting dilution. Monoclonal antibodies secreted from hybridomas3H11,1D7,2C9,1F8and3G3were identified to be subclass antibodies IgG2bK, IgG1κ, IgG2bK, IgG1κ and IgG1K. ELISA titers of the acsites were109,107,106,107and106, respectively. Indirect immunofluorescence assay (IFA) showed that the five mAb could combine with the natural CDV specifically. In western blotting, mAb1F8and3G3could react with protein N of CDV. Neutralization test indicated that mAb3H11and1D7had neutralizing ability to CDV, with the neutralizing titres of104and105. Addition ELISA revealed that the five mAb could recognize the different antigen epitopes of CDV.Part three:Establishment of the monoclonal antibodies-based sandwich ELISA for detecting canine distemper virusBased on the monoclonal antibodies against canine distemper virus (CDV), the sandwich ELISA was established to detect CDV. The purified monoclonal antibody (McAb)1D7was used to capture CDV antigen and McAb1F8conjugated with horseradish peroxidase (HRP) as trace antibody in the sandwich ELISA. The sandwich ELISA had no cross-reaction with canine parvovirus(CPV), canine parainfluenza virus (CPIV), canine adenovirus type-1(CAV-1) and canine coronvirs (CCV). The Sensitivity of the sandwich ELISA was102TCID5o/mL. The variation coefficients of the intra-assay and inter-assay were less5%. The sandwich ELISA was specific, sensitive and stable for the diagnosis of CD.Part four:Development of the monoclonal antibodies-based colloidal gold immunochromatography strip for the detection of canine distemper virusThe monoclonal antibodies-based colloidal gold immunochromatography strip was developed for the detection of canine distemper virus (CDV). The monoclonal antibody1F8(mAb1F8) conjugated with colloidal gold was used to capture CDV antigen and monoclonal antibody1D7(mAb1D7) to bind colloidal-gold McAb-CDV complexes at a test (T) line on the nitrocellulose strip. A downstream control (C) line of goat anti-mouse immunoglobulin G (IgG) antibody was used to capture excess free colloidal-gold conjugated1F8to validate test performance. The detection results indicated that the strip was specific to CDV and the detection limits of CDV was103TCID50/mL, which was equivalent to the sandwich ELISA established by using the same monoclonal antibodies1D7and1F8. The monoclonal antibodies-based colloidal gold immunochromatography strip is more convenient, rapid and simple for the clinical diagnosis of canine distemper (CD).Part five:Preparation of the neutralizing monoclonal antibody against canine distemper virusThe neutralization test of the five monoclonal antibodies (mAb) indicated that mAb3H11and1D7had neutralization ability to canine distemper virus (CDV), with the neutralizing titers of105and106, respectively.1D7was selected and made into biological product which had a significant effect on treating clinical CDV infected dogs.