Fluorescence immunological determination of immunoglobulin G in human serum by high-performance liquid gel-permeation chromatography |
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Authors: | H Hosotsubo K Arai Jun-Ichi Iwamura |
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Institution: | (1) Central Laboratory for Clinical Investigation, Osaka University Hospital, 1-1-50, Fukushima-ku, 553 Osaka, Japan;(2) Research Institute of Food Science, Kinki University, Kowakae, 577 Higashi-Osaka, Japan |
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Abstract: | Summary 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). |
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Keywords: | Column liquid chromatography Gel permeation Human serum IgG Fluorescent immune complex Fluoresence detection |
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