Evaluation of the fidelity of immunolabelling obtained with clone 5D8/1, a monoclonal antibody directed against the enteroviral capsid protein, VP1, ... View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2014-02

AUTHORS

Sarah J. Richardson, Pia Leete, Shalinee Dhayal, Mark A. Russell, Maarit Oikarinen, Jutta E. Laiho, Emma Svedin, Katharina Lind, Therese Rosenling, Nora Chapman, Adrian J. Bone, The nPOD-V Consortium, Alan K. Foulis, Gun Frisk, Malin Flodstrom-Tullberg, Didier Hober, Heikki Hyoty, Noel G. Morgan

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. More... »

PAGES

392-401

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/s00125-013-3094-7

DOI

http://dx.doi.org/10.1007/s00125-013-3094-7

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1016719188

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/24190581


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