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Clin Chim Acta. 2008 Jan;387(1-2):59-65. Epub 2007 Sep 8.

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

Author information

1
Department of Clinical Chemistry, University Hospital Maastricht, Maastricht, The Netherlands.

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.

PMID:
17904539
DOI:
10.1016/j.cca.2007.09.002
[Indexed for MEDLINE]

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