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Fluorescence immunological determination of immunoglobulin G in human serum by high-performance liquid gel-permeation chromatography

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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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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

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Key Words

  • Column liquid chromatography
  • Gel permeation
  • Human serum IgG
  • Fluorescent immune complex
  • Fluoresence detection