A high-performance liquid gel-permeation chromatographic method is described for the determination of human serum immunoglobulin G (IgG) by separating the fluorescent immuno complex from the free fluorescence-labeled antibody. Fluorescence-labeled antibody used in this study was fluorescein isothiocyanate (FITC)-labeled Fab fragment goat anti-human IgG (anti-IgG Fab). Immuno complexes and antibody of different molecular sizes can be separated. FITC-labeled anti-IgG Fab was added to the serum and the mixture is passed through the column. An immuno complex separates as well-delineated peak in the column void volume, and was measured by the fluorescence of the column eluate (Ex=490nm, Em=520nm). The total analysis time for a serum sample was approximately 15min. The minimum detection limit was 25 mg/dl. The relative standard deviation was below 2% (peak area). The results of the HPL-GPC analysis correlate well with those obtained by laser nephelometric assay (r=0.992).
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
M. E. Himel, P. G. Squire, J. Chromatogr.210, 443 (1981).
M. Flashner, H. Ramsden, L. T. Carne, Anal. Chem.135, 340 (1983).
T. Tomono, H. Ikeda, E. Tokunaga, J. Chromatogr.266, 39 (1983).
H. Hosotsubo, K. Arai, J. I. Iwamura, Chromatogr.23, 89 (1987).
H. Hosotsubo, K. Arai, J. I. Iwamura, J. Immunol. Methods85, 115 (1985).
H. Hosotsubo, K. Arai, J. I. Iwamura, J. Immunol. Methods98, 1 (1987).
R. L. Walsh, M. E. Coles, T. D. Geary, Clin. Chem.25, 151 (1979).
About this article
Cite this article
Hosotsubo, H., Arai, K. & Iwamura, J. Fluorescence immunological determination of immunoglobulin G in human serum by high-performance liquid gel-permeation chromatography. Chromatographia 25, 129–133 (1988). https://doi.org/10.1007/BF02259029
- Column liquid chromatography
- Gel permeation
- Human serum IgG
- Fluorescent immune complex
- Fluoresence detection