Development of a monoclonal antibody-based competitive enzyme-linked immunosorbent assay for detection of antibodies against duck adenovirus 3.

Abstract

BACKGROUND: Duck adenovirus 3 (DAdV-3), the emerging pathogen of a disease characterized by swelling and hemorrhage in liver and kidney, is widely distributed in China, resulting in serious economic losses to the duck farms. However, no vaccine is commercially available for the prevention of the disease. Thus, a precise and reliable diagnosis is of great value for the implementation of control measure to limit the spread of DAdV-3 infection. In this study, a competitive enzyme-linked immunosorbent assay (c-ELISA) was developed for detection of antibodies against DAdV-3. RESULTS: In the c-ELISA, a monoclonal antibody (mAb) 3D9, which was specific to Fiber-2 protein of DAdV-3, was labeled with horseradish peroxidase (HRP) and then used as secondary antibody. The cut-off value of the c-ELISA was determined as 18% using 48 clinical negative duck sera. The preliminary concordance evaluation based on 24 duck sera revealed that the results of the c-ELISA were highly consistent with those of indirect immunofluorescence assay (IFA). The cross-reactivity assessment showed that the c-ELISA only reacted with the positive sera against DAdV-3, but not with positive sera against other duck-associated viruses tested. The coefficients of variation of intra-batch and inter-batch assays were all below 10%, suggesting a good repeatability of the c-ELISA. Moreover, 133 duck sera from animal experiments in our laboratory were detected by c-ELISA and indirect ELISA. The results showed that the coincidence rate between the two assays was 90.98% (121/133) and the Kappa value was 0.815, revealing almost perfect agreement of the two assays. CONCLUSIONS: In summary, the c-ELISA developed here was specific, repeatable and reliable. The minimal cross-reactivity and independence of species-specific enzyme-conjugated secondary antibody made the c-ELISA a promising tool for the clinical diagnosis, serological epidemiological investigation, and evaluation of vaccine immunogenicity to control the disease caused by the infection of DAdV-3.