title: In vitro phage display in a rat beta cell line: a simple approach for the generation of a single-chain antibody targeting a novel beta cell-specific epitope.
abstract: OBJECTIVE
  The aim of the present study was to evaluate in vitro phage display in a beta cell line as a novel strategy for the isolation of beta cell-specific agents/biomarkers.
  
  
  METHODS
  A single-chain antibody (SCA) library was pre-incubated with AR42J cells in order to eliminate SCAs with exocrine binding properties. It was then panned against INS-1 cells to select beta cell-targeted antibodies.
  
  
  RESULTS
  By these means, we isolated a novel antibody, SCA B5, that binds rapidly (6.0 min) and with a 450-fold higher specificity to beta cells relative to exocrine cells. We estimated for SCA B5 a binding affinity in the low micromol/l range and 858 binding sites per beta cell. Confocal microscopy showed binding to the beta cell surface and confirmed subsequent internalisation. Moreover, staining of rat and human pancreatic tissue sections with SCA B5 suggests that the target epitope is presented in pancreatic beta cells of different origins. Infrared imaging revealed that labelling of beta cells with tracer SCA B5 is strictly dependent on beta cell mass. With competition assays we excluded insulin, glutamate decarboxylase, C-peptide and islet amyloid polypeptide as SCA B5 targets. In accordance with these predictions, SCA B5 homed in vivo highly selectively to normal beta cells and dysfunctional beta cells of diabetic rats. Moreover, accumulation of radioactively labelled SCA B5 in the pancreas was reduced by 80% after pre-injection with unlabelled SCA B5, thereby confirming the specific uptake in the pancreas.
  
  
  CONCLUSIONS
  We report a simple strategy for the generation of an SCA targeting a novel beta cell-specific epitope.
authors.affiliation: Department of Internal Medicine I, Division of Endocrinology and Metabolism, University Hospital Bergmannsheil, Ruhr-University Bochum, Bürkle de la Camp Platz 1, 44789, Bochum, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Ueberberg
authors.forename: D
authors.initials: D
authors.lastname: Ziegler
authors.forename: W
authors.initials: W
authors.lastname: Schechinger
authors.forename: J W
authors.initials: JW
authors.lastname: Dietrich
authors.forename: S
authors.initials: S
authors.lastname: Akinturk
authors.forename: H H
authors.initials: HH
authors.lastname: Klein
authors.forename: S
authors.initials: S
authors.lastname: Schneider
mesh_terms: D000818:Animals; D002460:Cell Line; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D007150:Immunohistochemistry; D050417:Insulin-Secreting Cells / Q000276:immunology*; D008856:Microscopy, Fluorescence; D019151:Peptide Library; D051381:Rats; D057127:Single-Chain Antibodies / Q000276:immunology*
chemical_list: D000939:Epitopes; D019151:Peptide Library; D057127:Single-Chain Antibodies