Human adenovirus (HAdV) infects the respiratory system, thus posing a threat to health. However, immunodiagnostic reagents for human adenovirus are limited. This study aimed to develop efficient diagnostic reagents based on monoclonal antibodies for diagnosing various human adenovirus infections. Evolutionary and homology analyses of various human adenoviral antigen genes revealed highly conserved antigenic fragments. The prokaryotic expression system was applied to recombinant penton, hexon, and IVa2 conserved fragments of adenovirus, which were injected into BALB/c mice to prepare human adenovirus-specific monoclonal antibodies. Enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and Western blotting were used to determine the immune specificity of the monoclonal antibodies. Indirect ELISA showed that monoclonal antibodies 1F10, 8D3, 4A1, and 9B2 were specifically bound to HAdV-3 and HAdV-55 and revealed high sensitivity and low detection limits for various human adenoviruses. Western blotting showed that 1F10 and 8D3 specifically recognized various human adenovirus types, including HAdV-1, HAdV-2, HAdV-3, HAdV-4, HAdV-5, HAdV-7, HAdV-21, and HAdV-55, and 4A1 specifically recognized HAdV-1, HAdV-2, HAdV-3, HAdV-5, HAdV-7, HAdV-21, and HAdV-55. IFAs showed that 1F10, 8D3, and 4A1 exhibited highly selective localization to A549 cells infected with HAdV-3 and HAdV-55. Finally, two antibody pairs that could detect hexon antigens HAdV-3 and HAdV-55 at low concentrations were developed. The monoclonal antibodies developed in this study show potential for detecting human adenoviruses.
Importance: In this study, we selected the three most conserved antigenic fragments of human adenovirus to prepare a murine monoclonal antibody for the first time, and human adenovirus antigenic fragments with heretofore unheard of degrees of conservatism were isolated. The three monoclonal antibodies with the ability to recognize human respiratory adenovirus over a broad spectrum were screened by hybridoma and monoclonal antibody preparation. Human adenovirus infections are serious; however, therapeutic drugs and diagnostic reagents are scarce. Thus, to reduce the serious consequences of human viral infections and adenovirus pneumonitis, early diagnosis of infection is required. The present study provides three monoclonal antibodies capable of recognizing a wide range of human adenoviruses, thereby offering guidance for subsequent research and development.
Keywords: ELISA; Western blot; hexon protein; human adenovirus; indirect immunofluorescence assay; monoclonal antibodies.