Development Of Monoclonal Antibodies Against Fiber2 Protein Of Serotype 4 Fowl Adenovirus And Their Application

Fowl Adenovirus(FAdV)belongs to adenovirus genus.Based on the sera cross-neutralization test and its genomic characteristics,FAdV is divided into 5 species(FAdV-A to FAdV-E)with 12 serotypes(1-7,8a,8b,9-11)respectively.Generally,FAdV infection only causes subclinical symptoms,while inclusion body hepatitis(IBH),hepatitis-hydropericardium syndrom(HPS),gizzard erosion and ulceration(GEU)are the main symptoms of acute infection.Since 2013,the outbreak of HPS caused by FAdV-4 has occurred frequently in most of the provinces in China,and the mortality of the diseased chickens can reach to 80%.The infection of FAdV-4 not only occurs in 3-4 week old broilers,but also in 10-20 week old layers,which has caused huge economic losses to poultry industry in China.However,commercial and specific diagnostics for FAdV-4 are still not available.In this study,FAdV-4 SD isolate was used to immunize Balb/c mice,and 4 monoclonal antibodies(mAbs)against Fiber2 protein(Fiber2)of FAdV-4 were generated.Based on these mAbs,a double monoclonal antibodies sandwich ELISA for detection of FAdV-4 was established in order to provide an effective and reliable detection technique for the rapid diagnosis of FAdV-4.1.Generation and characterization of monoclonal antibodies against Fiber2 protein of FAdV-4In order to obtain the monoclonal antibody against Fiber2 protein of FAdV-4,7 weeks old Balb/c mice were immunized with FAdV-4 SD isolate,and the purified GST-Fiber2 protein was used as the screening antigen.The hybridoma cell lines,which could stably secrete mAbs against Fiber2 protein of FAdV-4,were screened by indirect ELISA and designated as 3C2,4A3,5C4,and 5H6 respectively.The subclass of 3C2 and 5C4 was IgG2b,that for 4A3 was IgG2a,while the subclass of 5H6 was IgM,and light chain of these mAbs was all kappa chain.The ELISA titers of the four mAbs were higher than 1:160000.Specificity analysis showed that the four mAbs only reacted with FAdV-4 and its Fiber2 protein,but not with Fiber1 protein of FAdV-4 and the serotype 8 adenovirus(FAdV-8).Moreover,mAb 3C2 could not only neutralize the FAdV-4,but also efficiently immunoprecipitate the fiber-2 protein in LMH cells either infected with FAdV-4 or transfected with pcDNA3.1-Fiber2.The generation of the four mAbs against Fiber2 of FAdV-4 lays the foundation for developing rapid diagnostic kit and exploring the role of Fiber2 protein in the pathogenesis of FAdV-4 infection.2.Development of a double-mAb sandwich ELISA for detection of FAdV-4In order to develop a rapid and specific detection method for FAdV-4,three IgG subclass mAbs against Fiber2 protein of FAdV-4 were used in pairs to generate a double sandwich ELISA for detection of FAdV-4.In the ELISA,mAb 4A3 was used as a capture antibody and mAb 3 C2 labeled with HRP was used as a detection antibody.Specificity analysis showed that the ELISA generated here only reacted with FAdV-4,but not reacted with other avian pathogens including FAdV-8,Marek's disease virus(MDV),gosling plague virus(GPV),H9 avian influenza virus(H9N2),avian reticuloendodermis virus(REV),infectious laryngo tracheitis(ILTV)and avian leukosis virus(ALV).The limit of detection for the ELISA was 1.25 ng for the purified GST-Fiber2 protein and 1000 TCID50 for the FAdV-4 respectively.Moreover,the ELISA could be efficiently applied in detection of clinical samples from the diseased chickens.All these data demonstrated that the ELISA developed here provided a promising and specific approach for detection of FAdV-4 infection.