A specific immunoprecipitation method for isolating isoforms of insulin-like growth factor binding protein-3 from serum.
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.
Bons
Judith A P
JA
Department of Clinical Chemistry, University Hospital Maastricht, Maastricht, The Netherlands.
Michielsen
Etienne C H J
EC
de Boer
Douwe
D
Bouwman
Freek G
FG
Jaeken
Jaak
J
van Dieijen-Visser
Marja P
MP
Rubio-Gozalbo
M Estela
ME
Wodzig
Will K W H
WK
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*
D018972:Insulin-Like Growth Factor Binding Protein 3; D020033:Protein Isoforms