Immunohistochemical Detection of Enteroviruses in Pancreatic Tissues of Patients with Type 1 Diabetes Using a Polyclonal Antibody Against 2A Protease of Coxsackievirus

Introduction The need for antiserum for immunohistochemical detection (IHC) of enterovirus (EV) in formaldehyde fixed and paraffin-embedded (FFPE) samples is increasing. The gold standard monoclonal antibody (clone 5D8/1) against EV-envelope protein (VP1) was proven to cross-react with other proteins. Another candidate marker of EV proteins is 2A protease (2Apro ), which is encoded by EV gene and translated by the host cells during EV replication and participates processing proproteins to viral capsid proteins. Materials and methods We raised polyclonal antiserum by immunizing a rabbit with an 18-mer peptide of Coxsackievirus B1 (CVB1)-2Apro and examined the specificity and sensitivity for EV on FFPE tissue samples. Results ELISA study showed a high titer of antibody for 18-mer peptide of CVB1-2Apro , cross reacting with CVB3-2Apro peptide. IHC demonstrated that antiserum against 2Apro reacted with CVB1-infected and VP1-positive Vero-cells. Confocal laser scanning microscopy demonstrated that antigen stained by the 2Apro antibody located in the same cell with VP1 stained by 5D8/1. IHC using 2Apro antiserum showed dense staining in the islets of EV-associated fulminant type 1 diabetic pancreas and that located in the same cell stained positive for VP1 (5D8/1). Specificity of 2Apro antiserum by IHC staining was confirmed by negative 2Apro in 14 VP1-negative non-diabetic control pancreases. Conclusion Our study provides a new polyclonal antiserum against CVB1-2Apro which may be useful for IHC of EV-infected human tissues stored as archive of FFPE tissue samples.