A specific immunoprecipitation method for isolating isoforms of insulin-like growth factor binding protein-3 from serum

Clin Chim Acta. 2008 Jan;387(1-2):59-65. doi: 10.1016/j.cca.2007.09.002. Epub 2007 Sep 8.


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.

MeSH terms

  • Blotting, Western
  • Electrophoresis, Polyacrylamide Gel / methods
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Immunoprecipitation / methods*
  • Insulin-Like Growth Factor Binding Protein 3 / blood*
  • Protein Isoforms / blood*


  • Insulin-Like Growth Factor Binding Protein 3
  • Protein Isoforms