Immunohistochemical detection of enteroviruses in pancreatic tissues of patients with type 1 diabetes using a polyclonal antibody against 2A protease of Coxsackievirus

Aims/Introduction The need for antiserum for immunohistochemical (IHC) detection of enterovirus (EV) in formaldehyde-fixed and paraffin-embedded 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 (2A(pro)), which is encoded by the 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)-2A(pro), and examined the specificity and sensitivity for EV on formaldehyde-fixed and paraffin-embedded tissue samples. Results Enzyme-linked immunosorbent assay study showed a high titer of antibody for 18-mer peptide of CVB1-2A(pro), cross-reacting with CVB3-2A(pro) peptide. IHC showed that antiserum against 2A(pro) reacted with CVB1-infected and VP1-positive Vero cells. Confocal laser scanning microscopy showed that antigen stained by the 2A(pro) antibody located in the same cell with VP1 stained by 5D8/1. IHC using 2A(pro) antiserum showed dense staining in the islets of EV-associated fulminant type 1 diabetes pancreas and that located in the same cell stained positive for VP1 (5D8/1). Specificity of 2A(pro) antiserum by IHC staining was confirmed by negative 2A(pro) in 14 VP1-negative non-diabetes control pancreases. Conclusions Our study provides a new polyclonal antiserum against CVB1-2A(pro), which might be useful for IHC of EV-infected human tissues stored as archive of formaldehyde-fixed and paraffin-embedded tissue samples.