Preparation Of Rabbit Anti-goose IgY And Establishment Of Indirect ELISA Method For Novel Goose Astrovirus Antibody

Novel goose astrovirus disease is an acute and highly contagious disease caused by Novel Goose Astrovirus（NGAst V）,which is mainly characterized by gout in young geese.It mostly affects goslings between 4 to 21 days of age,and the mortality rate was up to30%,which caused a great blow to the development of goose industry.At present,the research on this disease mostly focuses on the detection of pathogenic nucleic acid and virus isolation and identification,but lacks serological research.In addition,enzyme-labeled secondary antibodies suitable for geese on the market are expensive and uneven in quality,affecting the accuracy of serological detection of the disease.With the increasing number of clinically inapparent infected goose flocks,a serological test is urgently needed in order to understand the distribution of anti-NGAst V antibody in goose flocks at different levels.Therefore,in this study,we prepared HRP-rabbit anti-goose IgY secondary antibody first,and based on this,an indirect ELISA method for detecting NGAst V antibody was established,which was applied to the detection of NGAst V antibody in geese of different ages in clinic,in order to provide an effective antibody detection method for the epidemiological investigation of NGAst V disease,as well as lays a foundation for the evaluation of vaccine efficacy in the future.1 Preparation of HRP Rabbit anti-goose IgY heavy chainFresh goose eggs were used to separate yolk,water-insoluble proteins in yolk were removed by water dilution method,lipoproteins were removed by ethanol fractionation precipitation method,and preliminarily purified goose yolk immunoglobulin（IgY）was obtained.Goose IgY was cut by SDS-PAGE electrophoresis to obtain goose IgY heavy chain protein with a molecular weight of 67 k Da～70 k Da,the goose IgY heavy chain protein concentration is 1.63 mg/m L.The goose IgY heavy chain protein was used to immunize rabbits according to the routine immunization program,and the rabbit serum was collected on the seventh day after the completion of the immunization procedure.The highest titer of serum measured by indirect ELISA test was 1:400,000;Western-blot identified that the serum could specifically bind to goose IgY heavy chain protein;rabbit anti-goose IgY serum was purified by Protein A-Sephrose FF affinity chromatography,freeze-dried and 2.5 mg of rabbit anti-goose IgY was taken for HRP labeling,and the HRP/Protein molar ratio was 2.18 as determined by spectrophotometer.2 Establishment of an indirect ELISA method for the detection of novel goose astrovirus antibodiesNGAstV ORF2 recombinant protein was used as coating antigen to establish an indirect ELISA for the detection of NGAst V antibodies.After optimization of the test conditions and parameters,the ELISA working conditions were determined as follows:NGAst V ORF2 recombinant protein was coated overnight at 4℃with a concentration of1.25μg/m L;5%skimmed milk was selected as the blocking solution and blocked at 37℃for 2 h;the dilution ratio of the primary antibody was 1:60 and incubated at 37℃for 2 h;the working concentration of HRP-rabbit anti-goose IgY heavy chain was 1:2000 and incubated at 37℃for 1 h;the TMB working conditions were 37℃and the reaction was 20min;and the cut-off value for positive judgment was OD₄₅₀≧0.159.The recombinant protein only reacted with NGAst V positive serum,was not cross-reacted with other virus positive serum such as goose parvovirus,goose Newcastle disease,goose tembusu;Sensitivity test of the method showed that the results of NGAst V positive serum were still positive at the highest dilution of 1:960;the coefficients of variation in the intra-batch repeatability test and inter-batch repeatability test were 1.838%～5.953%and 3.93%～6.572%,respectively,both within 10%.3 Preliminary application of indirect ELISA detection method for novel goose astrovirus antibodiesA total of 199 serum samples from 5 gosling hatchery forms and 2 goose farms（named A～G farms in turn）in Anhui Province were detected for NGAst V antibodies using the established indirect ELISA method and a total of 181 seropositive samples were detected,with a seroprevalence up to 90.95%（181/199）.Among them,A～D forms were 1day old gosling,the antibody positive rate was 76.92%-100%;F～G forms were 300 and360 day-old geese,the antibody positive rate was 94.12%to 96.67%.In summary,in this study,rabbit anti-goose IgY antibodies were prepared by immunizing rabbits with purified goose IgY heavy chain protein,and antibody purification and HRP labeling were performed to obtain HRP-rabbit anti-goose IgY antibodies;an indirect ELISA method for the detection of NGAst V antibodies was established using NGAst V ORF2 recombinant protein as the coating antigen,anti-NGAst V ORF2 goose serum as the primary antibody,and HRP-rabbit anti-goose IgY antibody as the secondary antibody,and 199 goslings and breeding geese serum were detected using this method,resulting in 76.92%to 100%positive rate of NGAst V antibodies.The establishment of this method will provide an effective means for the epidemiological investigation prevention and control of this disease.