TY - JOUR
T1 - Evaluation of the fidelity of immunolabelling obtained with clone 5D8/1, a monoclonal antibody directed against the enteroviral capsid protein, VP1, in human pancreas
AU - Richardson, Sarah J.
AU - Leete, Pia
AU - Dhayal, Shalinee
AU - Russell, Mark A.
AU - Oikarinen, Maarit
AU - Laiho, Jutta E.
AU - Svedin, Emma
AU - Lind, Katharina
AU - Rosenling, Therese
AU - Chapman, Nora
AU - Bone, Adrian J.
AU - Foulis, Alan K.
AU - Frisk, Gun
AU - Flodstrom-Tullberg, Malin
AU - Hober, Didier
AU - Hyoty, Heikki
AU - Morgan, Noel G.
N1 - Funding Information:
Funding This work was supported by funding from the European Union’s Seventh Framework Programme PEVNET (FP7/2007-2013) under grant agreement number 261441. Additional support was from a Diabetes Research and Wellness Foundation non-clinical research fellowship to SJR and from the Karolinska Institutet (KL), the Strategic Research Programme in Diabetes at the Karolinska Institutet (MF-T) and the Swedish Research Council (MF-T). The research was also performed with the support of the Network for Pancreatic Organ Donors with Diabetes (nPOD), a collaborative type 1 diabetes research project sponsored by the JDRF, and with a JDRF research grant awarded to the nPOD-V Consortium. Organ procurement organisations partnering with nPOD to provide research resources are listed at www.jdrfnpod.org/our-partners.php.
PY - 2014/2
Y1 - 2014/2
N2 - 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.
AB - 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.
KW - ATP5B
KW - Coxsackievirus
KW - Creatine kinase
KW - Dako 5D8/1
KW - Immunohistochemistry
KW - Islets of Langerhans
KW - Pancreatic beta cell
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U2 - 10.1007/s00125-013-3094-7
DO - 10.1007/s00125-013-3094-7
M3 - Article
C2 - 24190581
AN - SCOPUS:84893693165
VL - 57
SP - 392
EP - 401
JO - Diabetologia
JF - Diabetologia
SN - 0012-186X
IS - 2
ER -