The Estalisnment Of Colloidal Gold Immunochromatography Detection Method For Canine Parvovirus

Canine parvovirus disease(CPVD)is a kind of highly contagiously disease caused by canine parvovirus(CPV),the sick dogs are characteristics of severe vomiting,hemorrhagic enteritis and myocarditis.The disease spread so fast that it has high incidence and high mortality.It can disease all over the year,but mostly in the spring and summer.Furthermore,CPVD has brought great harm to canine fanning.So it is significant to establish rapid,specific and simple detection methods for the prevention of CPVD.In this study,we preparated to establish sandwich ELISA and colloidal gold immunochromatographic assay for detecting canine parvovirus,And try to apply the Biotin-Avidin system to the colloidal gold immunochromatographic strip in order to improve the sensitivity of the test strip.The paper contains three parts:1.Development of the sandwich ELISA with monoclonal antibodies for canine parvovirus detectionBased on the monoclonal antibodies against canine parvovirus(CPV)which prepared in laboratory,the sandwich ELISA was developed for the detection of CPV.The purified monoclonal antibody 3A5(mAb-3A5)was respectively coated to catch CPV-2 and labeled with horseradish peroxidase(HRP)to detect antigen in sandwich ELISA.The reaction condition was optimized and the results showed that the sandwich ELISA was non-reactive with positive serum of other virus that canine were easy to infect.It is sensitive for sandwich ELISA to detect at least 102TCID50/mL.The repetitive experiment indicate the variation coefficient of intra-assay and inter-assay were less 7%.The sandwich ELISA had good specificity,sensitivity and repeatability for the diagnosis of CPVD.2.Preparation for the colloidal gold immunochromatography strip with monoclonal antibody for the detection of canine parvovirusBased on the sandwich ELISA,the colloidal gold immunochromatography strip was developed to detect of canine pavovirus(CPV).The colloidal gold was labled mAb-3A5 to catch CPV antigen.The mAb-3A5 and goat anti-mouse immunoglobulin G(IgG)antibody were respectively coated at test(T)line and control(C)line to bind colloidal-gold mAb-3A5 complexes on the nitrocellulose strip.The reaction condition was optimized and the experiment results showed that it was specific,sensitive,repeatable and stable of colloidal gold immunochromatography strip for the clinical detection of CPV,and it had no significant difference with the detection results of sandwich ELISA based on the same monoclonal antibody.The colloidal gold immunochromatography strip based on mAb-3A5 was more convenient,rapid and sensitive for the clinical detection of CPV.3.Application of biotin-avidin in the colloidal gold immunochromatography stripBased on the colloidal gold immunochromatographic technology,the signal amplification of biotin-avidin system(BAS)was applied to the detection of canine pavovirus(CPV).The mAb-3A5 labeled with BNHS was used to catch CPV antigen and streptavidin combined with colloidal gold to bind BNHS mAb-3A5 complexes.The mAb-3A5 and goat anti-mouse immunoglobulin G(IgG)antibody worked separately at test(T)line and control(C)line to capture colloidal-gold complexes to validate test performance.The results showed that the BNHS was successfully labled with mAb-3A5,and obtained colloidal gold-streptavidin,but the preparation of the test strip still needs to be further optimized.So we need fuether experiment to apply the signal amplification of BSA to the colloidal gold immunochromatography strip.