Preparation Of Monoclonal Antibody Against Canine Coronavirus M Protein And Its Application On Colloidal Gold Test Strip

Canine Coronavirus(CCoV)is an enveloped,single-stranded,positive-sense RNA virus.CCoV infection can cause typical clinical symptoms of puppy gastroenteritis,such as loss of appetite,diarrhea and vomiting,with high morbidity and mortality rate.The full length genome of the CCoV is about 30 kb,and the 3’ end encodes 4 structural proteins,namely,spiking protein(S),envelope protein(E),membrane protein(M)and nucleocapsid protein(N).M protein is a membrane-structure glycoprotein,which is the most abundant among envelope proteins,and is a key component of virus assembly and morphogenesis.It contains B cell recognition epitopes.The gene encoding M protein is highly conserved and has low homology with the M gene of coronaviruses within other genera.Therefore,M protein has become one of the main targets for detection and diagnosis of canine coronavirus.Canine gastroenteritis can be caused by a variety of pathogens,including viruses,bacteria or parasites.In addition to CCoV,canine parvovirus(CPV),canine adenovirus type 2(CAV-2)and canine distemper virus(CDV)are the most common viral enteritis pathogens.Therefore,rapid virological detection is essential for the control of canine viral enteritis.In order to develop suitable colloidal gold immunochromatographic test strip for rapid diagnosis of canine coronavirus,two hybridioma cell lines were prepared and screened in this study that produce canine coronavirus M protein-specific monoclonal antibody.The purification methods of different monoclonal antibody IgG subclass were compared,and the preparation process of colloidal gold immunochromatographic strip for canine coronavirus was established.The strip has good specificity and high sensitivity.This thesis contains three parts:1.Preparation and biological characterization of monoclonal antibody to M and N protein of canine coronavirusIn order to produce monoclonal antibody specific to canine coronavirus(CCoV),BALB/C mice were immunized with purified CCoV antigen for 4 times.Spleen cells from immunised mouse and SP2/0 cells were fused with polyethylene glycol(PEG),and antibody-positive hybridoma cells were screened by indirect immunofluorescence assay(IFA).Two hybridioma cell lines,named 2H11 and 2G3,which can secrete monoclonal antibodies against CCoV M protein stably,were obtained by three times of cloning.The antibody titers were stable in vitro for 40 consecutive generations.Characterization of mAb immunoglobulin(Ig)subclass showed that 2H11 mAb was IgG3 subclass and 2G3 mAb was IgGl subclass.Western blot and IFA analysis showed that 2H11 and 2G3 mAb specifically recognized CCoV M protein(28-32 kDa).The IFA titers of 2H11 and 2G3 hybridioma culture supernatants were 1:1000,and the ELISA titers were≥1:105.Monoclonal antibody specificity test showed that these monoclonal antibodies had no cross-reactivity with canine distemper virus(CDV),canine parvovirus(CPV),canine adenovirus(CAV),or canine parainfluenza virus(CPIV).The establishment of hybridoma cell line with specific monoclonal antibody against CCoV M protein has laid a solid foundation for the identification of CCoV and the establishment of related diagnostic methods.2.Comparative study on methods of purification of mouse ascites monoclonal antibodyIn order to purify the monoclonal antibody in mouse ascites,mouse ascites of two hybridiomas,2G3 and 2H11,were prepared in three batches.The two strains of mAb were purified by saturated ammonium sulfate precipitation method,caprylic acid-ammonium sulfate method,affinity chromatography and ultracentrifugation-dialysis method,respectively.The protein concentration,purity,recovery rate and the antibody titer were analyzed and compared.The results showed that different Ig subclasses of monoclonal antibodies necessitated different purification methods.The optimal purification method of 2G3 monoclonal antibody(IgG1 subclass)was affinity chromatography,which had a low recovery rate but high purity.Because octanoic acid treatment damages IgG3,and the affinity column of protein G does not tightly bound to IgG3 during affinity chromatography,octanoic acid-ammonium sulfate and affinity chromatography are not suitable for the purification of 2H11 mAb.The optimal purification method of 2H11 mAb was ultracentrifugation-dialysis.3.Preparation of colloidal gold immunochromatography strip for detection of canine coronavirusIn order to prepare colloidal gold immunochromatography strip,20 nm colloidal gold particles were prepared by trisodium citrate reduction method.The monoclonal antibody 2G3 was selected as the gold label antibody to label the colloidal gold.After optimization,the optimal pH of gold label was 9.0,and the optimal protein concentration was 100 μg/mL.Monoclonal antibody 2H11 was used as the detection antibody and was coated on the NC membrane as the detection line(T line)at 1mg/mL.Rabbit anti-rat IgG was used as the quality control antibody and was coated as the quality control line(C line)at lmg/mL.The specificity test showed that the strip had no cross-reactivity with CDV,CAV,CPIV and CPV.The sensitivity test showed that the virus could be detected at 102.8TCID50/mL.For clinical fecal samples,the coincidence rate between the lab-prepared strip and the imported commercial strip was 100%.In this study,the colloidal gold immunochromatographic strip for detecting CCoV was successfully prepared,which has important application value for early diagnosis,prevention and control of CCoV.