Identification Of Monoclonal Antibodies Epitope And Development Of Commom ELISA Against Group Ⅰ Fowl Adenovirus

Fowl adenoviruses（FAdVs）are classified in the family Adenoviridae,genus Aviadenovirus,is an important infectious disease pathogen endangering the poultry industry.FAdV-I infection have been reported worldwide,mainly caused hydropericardium syndrome（HPS）,inclusion body hepatitis,and gizzard erosion.And since 2015,outbreaks of HPS caused by a novel genotype of FAdV-4 infection have caused serious economic losses in China.The development of simple and rapid FAdVs antigen and antibody diagnosis method is essential for disease prevention and control.As previous,a novel FAdV-4 HLJFAd15 strain was isolated and identified,determined the whole genome sequence,and pathogenicity experiment was studied in SPF chickens.Based on previous research,this study prepared ten monoclonal antibodies（mAbs）against the capsid hexon protein of HLJFAd15 and identified B-cell epitopes of FAdVs.Furthermore,we developed a common ELISA for detecting antibodies against FAdV-I using puritied virions of HLJFAd15.The diagnostic methods of FAdVs mainly rely on PCR and DNA sequencing.In order to further development FAdVs pathogenic detection methods,we prepared ten mAbs against FAdV-4 Hexon L1region（230 amino acids）.Epitope analysis of five mAbs was performed by truncated expression.Finally,three B-cell epitopes(³¹PLAPKESMFN⁴⁰,⁷⁹KISGVFPNP⁸⁷,¹⁸¹DYDDYNIGTT¹⁹⁰)on the L1-hexon of FAdV-4 were identified.Furthermore,a common B-cell epitope(³¹PLAPKESMFN⁴⁰)for all species FAdVs and two FAdV-C-specific epitopes(⁷⁹KISGVFPNP⁸⁷ and ¹⁸¹DYDDYNIGTT¹⁹⁰)were identified by bioinformatics tools and IFA assay.In this study,ten mAbs were successfully prepared,two FAdV-C specific mAbs and a FAdV-I universal mAb were selected,which laid the foundation for further development of FAdVs antigen detection methods and differential diagnostic reagent.ELISA detection method with simplicity,high sensitivity and strong specificity,was widely used in large-scale serological investigation and vaccine evaluation.Many serotypes of highly pathogenic FAdVs have been reported around the world.Unfortunately,there is currently no commercial FAdV-I ELISA kit in China.Based on cross-reactivity of FAdVs,we purified HLJFAd15 virus particles,developed a common ELISA for detecting antibodies against serotypes of FAdV-I.Specificity assay showed the developed ELISA was able to distinguish between antibodies against FAdV-I,FAdV-III and other heterologous viruses without any cross-reaction,and could simultaneously detect FAdV-1,FAdV-4,FAdV-8a,FAdV-9,FAdV-10,FAdV-11 positive sera in FAdV-I.The developed ELISA showed higher sensitivity than the FAdV-1 based ELISA for detecting antibodies against the novel emergent FAdV-4.This study has developed a common ELISA antibody detection method for FAdV-I which has strong specificity and highly sensitivity,could provide a powerful tool for seroepidemiological investigation and FAdVs vaccine development.