title: The use of phage display to distinguish insulin autoantibody (IAA) from insulin antibody (IA) idiotypes.
abstract: OBJECTIVE
  Radiobinding assays (RBA) are unable to differentiate insulin autoantibodies (IAA) from insulin antibodies (IA). We sought to establish whether random peptide phage display might generate reagents with which to distinguish IAA idiotopes from IA idiotopes.
  
  
  METHODS
  Two insulin-binding sera were used to select phagotopes from a phage library. The first, designated IAS, came from an insulin-treated patient with the insulin autoimmune syndrome, and was known to contain both IA and a high titre of human insulin specific (B30 threonine dependent) IAA. The second, designated IDD, was taken from a newly-diagnosed IAA(+) Type 1 diabetic patient. Phage colonies selected by insulin-purified IgG extracts of IAS and IDD were selected at random for DNA sequencing, and tested for their reactivity with insulin antibodies and ability to distinguish disease-associated idiotopes.
  
  
  RESULTS
  Seven phagotopes bound IAS and the phagotope designated IAS-9, corresponding to sequence KRSRLDV, gave the highest binding standard deviation (SD) score. Seven phagotopes bound IDD and the phagotope designated IDD-10, corresponding to sequence LGRGGSK, bound most strongly. IAS-9 was able to displace insulin binding in IAS and all of ten insulin-treated Type 2 diabetic patients, but not the IAA present in any of the eight patients with newly-diagnosed Type 1 diabetes. IDD-10, on the other hand, could displace insulin binding detected in the sera of eight patients with untreated Type 1 diabetes (IAA), but not in IAS or sera of the insulin-treated Type 2 diabetics.
  
  
  CONCLUSIONS
  Phagotopes provide reagents which between them can distinguish positively as well as negatively diabetes-associated IAA from non-diabetes associated IAA and from IA.
authors.affiliation: Department of University Medicine, Plymouth Campus, United Kingdom. devasenan.devendra@uchsc.edu
authors.forename: D
authors.initials: D
authors.lastname: Devendra
authors.forename: T S
authors.initials: TS
authors.lastname: Galloway
authors.forename: S J
authors.initials: SJ
authors.lastname: Horton
authors.forename: A
authors.initials: A
authors.lastname: Evenden
authors.forename: U
authors.initials: U
authors.lastname: Keller
authors.forename: T J
authors.initials: TJ
authors.lastname: Wilkin
mesh_terms: D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000097:blood*; D006801:Humans; D007130:Immunoglobulin Idiotypes / Q000097:blood*; D007328:Insulin / Q000097:blood / Q000235:genetics* / Q000276:immunology*; D007330:Insulin Antibodies / Q000097:blood*; D010446:Peptide Fragments / Q000737:chemistry; D019151:Peptide Library
chemical_list: D001323:Autoantibodies; D007130:Immunoglobulin Idiotypes; D007328:Insulin; D007330:Insulin Antibodies; D010446:Peptide Fragments; D019151:Peptide Library