title: A specific immunoprecipitation method for isolating isoforms of insulin-like growth factor binding protein-3 from serum.
abstract: BACKGROUND
  This report describes an in-house developed immunoprecipitation method to isolate insulin-like growth factor binding protein-3 (IGFBP-3) and its isoforms from serum. The method was compared to other existing immunoprecipitation methods. The study of IGFBP-3 isoforms is relevant for further studies on congenital defects in glycosylation (CDG), galactosemia, and alcoholic liver cirrhosis.
  
  
  METHODS
  Monoclonal and/or polyclonal anti-human IGFBP-3 antibodies were covalently immobilised on protein-A Sepharose beads using dimethyl pimelimidate as cross-linker. By incubation with these immobilised antibodies, intact IGFBP-3 and fragments of IGFBP-3 were isolated from serum. Enzyme-linked immunosorbent assay (ELISA) and one-dimensional gel electrophoresis (1-DE) experiments were performed to define the optimal immunoprecipitation method. Isolated proteins were separated by 1-DE and two-dimensional gel electrophoresis (2-DE) and visualised by Western blotting.
  
  
  RESULTS
  ELISA and 1-DE results illustrated that an optimal isolation was performed using PBS for the incubation with serum. Laemmli sample buffer, containing 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride and sodium dodecyl sulfate, or urea/CHAPS was optimal for the elution. Clinical validation was performed using CDG-Ia serum samples. The 2-DE experiments showed characteristic isoform patterns for CDG-Ia.
  
  
  CONCLUSIONS
  The optimized in-house developed immunoprecipitation method resulted in specific detection of IGFBP-3 isoforms and is suitable for further studies on glycosylation defects.
authors: Department of Clinical Chemistry, University Hospital Maastricht, Maastricht, The Netherlands.
authors: Judith A P
authors: JA
authors: Bons
authors: Etienne C H J
authors: EC
authors: Michielsen
authors: Douwe
authors: D
authors: de Boer
authors: Freek G
authors: FG
authors: Bouwman
authors: Jaak
authors: J
authors: Jaeken
authors: Marja P
authors: MP
authors: van Dieijen-Visser
authors: M Estela
authors: ME
authors: Rubio-Gozalbo
authors: Will K W H
authors: WK
authors: Wodzig
mesh_terms: D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000097:blood*; D020033:Protein Isoforms / Q000097:blood*
chemical_list: D018972:Insulin-Like Growth Factor Binding Protein 3; D020033:Protein Isoforms