Establishment Of Canine Parvovirus Sandwich ELISA And Colloidal Gold Immunochromatographic Assay

Canine parvovirus,unlike the Minute virus of canine(also known as CPV-1)discovered in 1967,was named Canine Parvovirus Type 2(CPV-2)in order to distinguish the two.CPV-2 is a DNA virus,which does not have a polymerase nor encodes such an enzyme,and must use the cell's DNA polymerase ? to synthesize viral DNA.The test proved that the virus has a defective replication ability.When culture in vitro,virus must be inoculated at the same time or at the latest within 24 hours to achieve good virus proliferation.Canine parvovirus disease poses a serious threat to the healthy development of the dog industry.There was also report that small pandas are infected with CPV-2 and panda-origin canine parvoviruses,indicating that the susceptible host range of the virus is expanding.The incidence and mortality of canine parvovirus disease is high,and the cure rate is low.No specific drug has yet been developed in clinical practice,and the current use of the vaccine strain may differ from the amino acid sequence of the variant virus strain,increasing the probability of immune failure and misdiagnosis.Early diagnosis is of great significance for the prevention and treatment of the disease.The colloidal gold test strip is widely used,due to it is easy to operate and the test result is intuitive.widely used in pet disease detection.At present,the commercially available colloidal gold test strip is mainly manufactured by Anign Corporation of South Korea.The test strip has good sensitivity and specificity,but its price is high,and the production process is protected by patents.The purpose of this study was to prepare monoclonal antibodies with good specificity and high affinity.The sandwich ELISA and colloidal gold immunochromatographic assays based on this method were established to improve the sensitivity of the diseasediagnosis and develop test strips with independent intellectual property rights provide.The test is as follows:1.Preparation of canine parvovirus monoclonal antibody hybridoma cell lineUsing feline kidney cells(FK81)to culture virus.The virus was purified by sucrose density gradient centrifugation.Five BALB/c mice were immunized with the purified virus and selected for intraperitoneal injection.The spleen cells fusing with myeloma cells by PEG4000.After screening and subcloning,3 hybridoma cell strains of monoclonal antibodies against canine parvovirus were obtained,named 1H3,2B5,and 1B1.The Ig isotypes of mAbs were IgG1??IgM? and IgG1?.Three monoclonal antibodies ascites titer were:102,102 and 106;and the antibody titer of cell culture supernatant was 24,23 and 210.The results of indirect immunofluorescence test showed that the three monoclonal antibodies could be specifically associated with the FK81 infection of CPV-2,and the green fluorescence could be observed.The neutralization titers of mAbs ascites were determined by the fixed virus dilution serum method,1H3:103,2B5:102 and 1B1:1062.Establishment of the monoclonal antibody sandwich ELISA for detection of canine parvovirusThe purified monoclonal antibody 1B1 of canine parvovirus is used as a capture antibody,monoclonal antibody 1H3 labeled by horseradish peroxidase as a detection antibody.The optimal detection conditions are:the package concentration of 1B1 was 2?g/mL,the blocking conditions were PBST(phosphate buffer containing 0.05%Twain 20)containing 5%non-fat milk powder blocked at 37? for 2 hours,the antigen is best incubated at 37? for 1 hour,the most suitable dilution multiple of HRP-1H3 is 1:4000.The established ELISA method detected canine parainfluenza virus(CPIV),canine adenovirus type-1(CAV-1),canine distemper virus(CDV),canine coronavirus(CCV),the results were all negative,indicating that this method has good specificity.The repetitive experiment indicate the variation coefficient of intra-assay and inter-assay were less 10%.The sensitivity of the sandwich ELISA was 102 TCID50/mL.3.Development of colloidal gold immunochromatography test paper for detection of canine parvovirusBased on the CPV-2 sandwich detection method established in the second chapter of the test,assembled the immunocolloid gold test paper strips.The 1H3 was enveloped on the colloidal gold surface to form an immuno colloid gold,the best pH environment is 7.0,the detection antibody is 1B1,the quality control antibody is the goat anti-mouse IgG.The protein concentration of 1H3 is 18 ?g/mL,the best concentration of 1B1 is 1.5 mg/mL,the goat anti-mouse IgG is 1.0 mg/mL.Use the test paper to detected four common canine viral diseases(CPIV,CAV-1,CDV,CCV),the results were negative,use the test strips which were made in the same batch and in different batches to diagnosis CPV-2,the test paper has good repeatability,the detection limits of CPV-2 was 102 TCID50/mL.Detection of 30 clinical samples by using test paper,the results were the same with PCR.Colloidal gold test strip can be used for the detection of CPV-2.