title: Evaluation of the fidelity of immunolabelling obtained with clone 5D8/1, a monoclonal antibody directed against the enteroviral capsid protein, VP1, in human pancreas.
abstract: OBJECTIVE
  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
  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.
authors.affiliation: Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, RILD Building, Barrack Road, Exeter, EX2 5DW, UK, S.Richardson@exeter.ac.uk.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Richardson
authors.forename: Pia
authors.initials: P
authors.lastname: Leete
authors.forename: Shalinee
authors.initials: S
authors.lastname: Dhayal
authors.forename: Mark A
authors.initials: MA
authors.lastname: Russell
authors.forename: Maarit
authors.initials: M
authors.lastname: Oikarinen
authors.forename: Jutta E
authors.initials: JE
authors.lastname: Laiho
authors.forename: Emma
authors.initials: E
authors.lastname: Svedin
authors.forename: Katharina
authors.initials: K
authors.lastname: Lind
authors.forename: Therese
authors.initials: T
authors.lastname: Rosenling
authors.forename: Nora
authors.initials: N
authors.lastname: Chapman
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Bone
authors.forename: Alan K
authors.initials: AK
authors.lastname: Foulis
authors.forename: Gun
authors.initials: G
authors.lastname: Frisk
authors.forename: Malin
authors.initials: M
authors.lastname: Flodstrom-Tullberg
authors.forename: Didier
authors.initials: D
authors.lastname: Hober
authors.forename: Heikki
authors.initials: H
authors.lastname: Hyoty
authors.forename: Noel G
authors.initials: NG
authors.lastname: Morgan
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000956:Antigens, Viral / Q000378:metabolism; D015153:Blotting, Western; D036022:Capsid Proteins / Q000276:immunology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D003429:Cross Reactions; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology / Q000378:metabolism* / Q000821:virology; D004770:Enterovirus / Q000378:metabolism*; D004769:Enterovirus Infections / Q000150:complications / Q000276:immunology / Q000378:metabolism*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D050417:Insulin-Secreting Cells / Q000378:metabolism; D008297:Male; D010179:Pancreas / Q000276:immunology / Q000378:metabolism* / Q000821:virology; D015203:Reproducibility of Results; D014779:Virus Replication
chemical_list: D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D036022:Capsid Proteins