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. 2014 Feb;57(2):392-401. doi: 10.1007/s00125-013-3094-7. Epub 2013 Nov 5.


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.

Publication types

  • Evaluation Study

MeSH terms

  • Antibodies, Monoclonal / metabolism*
  • Antigens, Viral / metabolism
  • Blotting, Western
  • Capsid Proteins / immunology*
  • Cell Proliferation
  • Cells, Cultured
  • Cross Reactions
  • Diabetes Mellitus, Type 1 / immunology
  • Diabetes Mellitus, Type 1 / metabolism*
  • Diabetes Mellitus, Type 1 / virology
  • Enterovirus / metabolism*
  • Enterovirus Infections / complications
  • Enterovirus Infections / immunology
  • Enterovirus Infections / metabolism*
  • Female
  • Humans
  • Immunohistochemistry
  • Insulin-Secreting Cells / metabolism
  • Male
  • Pancreas / immunology
  • Pancreas / metabolism*
  • Pancreas / virology
  • Reproducibility of Results
  • Virus Replication


  • Antibodies, Monoclonal
  • Antigens, Viral
  • Capsid Proteins