Preparation And Identification Of Monoclonal Antibodies Against Goose Parvovirus And Development Of Colloidal Gold-based Immunochromatographic Assay For Rapid Detection

Goose parvovirus(GPV),also known as gosling plague virus,which is an acute or subacute sepsis in 4 to 20 days-old goslings characterized by high infectiousness and exudative inflammatory bowel disease.In 1956,the disease was first discovered and named by Fang Dingyi,a Chinese scholar,then GPV was reported by various countries in the world.Goose parvovirus belongs to the family Parvoviridae,and the virus belongs to the single-stranded linear DNA virus.The GPV particles can be observed in hexagonal or circular appearance with a diameter of 20 nm through scanning electron microscope.The virus has three structural proteins,of which the VP3 protein is the major capsid protein and contains the major antigenic determinants of the GPV.To study the structure and function,of goose parvovirus and establish a rapid and effective detection method is an effective measure to control the disease.This study successfully prepared anti-GPV monoclonal antibodies,established a colloidal gold-based immunochromatographic assay for rapid detection of goose parvovirus,and provided technical support for the prevention and treatment of the disease.The content of this study was divided into the following two parts:1.Preparation of anti-GPV monoclonal antibodiesIn this study,GPV viral fluid was obtained through inoculated 11-day-old goose embryos for extensive entoxication,allantoic fluid was taken and the virus was concentrated by ultracentrifugation.The concentrated allantoic fluid was further purified by sucrose density gradient centrifugation and the purified GPV virus fluid was obtained.Purified GPV virus fluid was used as an immunogen to immunize BALB/c mice at 6-8 weeks of age,and positive clones were screened by cell fusion and ELISA.Finally,three hybridoma cell lines which could stably secrete monoclonal antibodies were obtained.Hybridoma cell lines respectively named A2D2,B5D2,and C3B2,and the latter two hybridoma cell lines was detected for related characterization with B5D2 and C3B2.The subtypes of the B5D2 and C3B2 antibodies were identified as IgG1 by the monoclonal antibody subtype identification kit.The titer of cell supernatants of both monoclonal antibodies was 1:100 and the potency of induced ascites was 1:128000 and 1:64000 respectively.The results of indirect ELISA showed that the two monoclonal antibodies only reacted with GPV and had no cross-reactivity with AIV,DHV,NDV and other viruses.IFA results showed that both monoclonal antibodies reacted specifically with the purified GPV virus and goose parvovirus.The successful development of anti-GPV monoclonal antibodies lays the foundation for the immunological diagnosis and pathogenic mechanism research of GPV.2.Development and application of colloidal gold-based immunochromatographic assay forrapid detectionIn this study,we first immunized healthy rabbits by mixing laboratory-preserved VP3 recombinant protein with Freund's adjuvant.After 4 immunizations,blood was collected two months later and serum was prepared,and serum antibody titer was detected by ELISA.Anti-GPV polyclonal antibody was purified from the serum by sequential precipitation with caprylic acid and ammonium sulphate,and the antibody titer could reach 1:1000 detected by ELISA.The colloidal gold was prepared by the trisodium citrate reduction method,and the optimal concentration of the monoclonal antibody to be labeled was determined to be 9?g/mL.Using anti-GPV monoclonal antibody B5D2 prepared as the capture antibody and the mouse anti-VP3 protein polyclonal antibody prepared as the coating antibody,the colloidal gold immunochromatographic test strip was successfully established to detect goose parvovirus.The purified GPV suspension which concentrated in the previous work was diluted to different concentrations,and ICG strip test,agar diffusion test and PCR test were carried out respectively with different concentration solutions.The results showed that the lowest limit of detection of the ICG strip test was 1.2 ?g/mL.Although its sensitivity slightly lower than PCR test,it is 100 times higher than the agar diffusion test.Goose parvovirus and other goose samples including avian influenza virus(H9),muscovy duck parvovirus,duck hepatitis virus,duck plague virus,tembusu virus,fowl adenovirus,goose reovirus and Avian Paramyxovirus were examined by the immunochromatographic strip,and the test results show that the immunochromatographic strip had high specificity for detecting GPV,and no cross-reaction was observed.To determine the stability of the immunochromatographic strip,the ICG assay was carried out with the same batch of strips after stored at 4? and 25? for 8 months,and the results showed that there was no significant decrease for detection effect.ICG strips test established in the study were performed on 92 clinical samples of suspected cases of GPV,and comparing the results of these tests with those obtained via agar diffusion test and PCR test.The results showed that the positive rate of goose allantoic fluid was determined to be 88.8%by the ICG strips test and 92.6%by the PCR test,and the coincidence rate of these two methods was 96%.For supernatants of tissue homogenate,it was 84.2%by the ICG strips test and 97.3%by the PCR test,and the coincidence was only 76.4%.For all clinical samples,the coincidence rate was up to 91.9%.Moreover,only few positive samples could be detected by agar diffusion test.The detection method of colloidal gold-based immunochromatographic strips for rapid detection of GPV established in this study has good specificity,sensitivity and stability,and provides a new method for the rapid diagnosis of goose parvovirus.