Evaluation of the fidelity of immunolabelling obtained with clone 5D8/1, a monoclonal antibody directed against the enteroviral capsid protein, VP1, in human pancreas

Richardson, S. J. et al. (2014) Evaluation of the fidelity of immunolabelling obtained with clone 5D8/1, a monoclonal antibody directed against the enteroviral capsid protein, VP1, in human pancreas. Diabetologia, 57(2), pp. 392-401. (doi: 10.1007/s00125-013-3094-7) (PMID:24190581)

Abstract

Aims/hypothesis Enteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes.

Methods Enteroviral VP1, CKB and ATP5B expression were analysed by western blotting, RT-PCR and immunocytochemistry in a range of cultured cell lines, isolated human islets and human tissue.

Results Clone 5D8/1 labelled CKB, but not ATP5B, on western blots performed under denaturing conditions. In cultured human cell lines, isolated human islets and pancreas sections from patients with type 1 diabetes, the immunolabelling of ATP5B, CKB and VP1 by 5D8/1 was readily distinguishable. Moreover, in a human tissue microarray displaying more than 80 different cells and tissues, only two (stomach and colon; both of which are potential sites of enterovirus infection) were immunopositive when stained with clone 5D8/1.

Conclusions/interpretation When used under carefully optimised conditions, the immunolabelling pattern detected in sections of human pancreas with clone 5D8/1 did not reflect cross-reactivity with either ATP5B or CKB. Rather, 5D8/1 is likely to be representative of enteroviral antigen expression.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Foulis, Dr Alan
• Authors: Richardson, S. J., Leete, P., Dhayal, S., Russell, M. A., Oikarinen, M., Laiho, J. E., Svedin, E., Lind, K., Rosenling, T., Chapman, N., Bone, A. J., Foulis, A. F., Frisk, G., Flodstrom-Tullberg, M., Hober, D., Hyoty, H., and Morgan, N. G.
• College/School: College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
• Journal Name: Diabetologia
• Publisher: Springer-Verlag
• ISSN: 0012-186X
• ISSN (Online): 1432-0428