Fluorescein Isothiocyanate-Labeled Protein G as an Affinity Ligand in Affinity/Immunocapillary Electrophoresis with Fluorescence Detection

authored by: Oscar Werner Relf, Ruth Freitag, Ralf Lausch, Thomas Scheper
Abstract: Antibodies from human sera (h-IgG) were tagged with a fluorescent dye (fluorescein isothiocyanate, FITC) through the affinity reaction of FITC-labeled protein G with the Fc fragment of the antibodies. The complexes were quantified by capillary zone electrophoresis (CZE) within 1 min, i.e., fast enough to prevent their dissociation during the measurement. Conditions for the affinity reaction and the CZE analysis could thus be optimized independently. When an FITC-labeled protein G concentration of 10⁻⁶ mol/L was used, h-IgG concentrations between 10⁻⁶ and 10⁻⁹ mol/L were reproducibly quantified (STD < 2%), using an LIF detector. A correlation coefficient, r², of 0.9988 was established between the peak height and the IgG concentration. Alternatively, h–IgG containing serum samples and the FITC-labeled protein G were simply injected into the CE capillary in consecutive zones, followed by the application of the electrical field. Within 2 min, the affinity complexes were resolved and the IgG content of the serum quantified (r² = 0.9986). The injection sequence was of no consequence. The measurements agreed well with those found in a single radial immunodiffusion (SRID) assay. In addition FITC-labeled protein G-tagged anti-h-IgG₁ antibodies were used to detect the specific antigen of the involved antibody, namely, h-IgG₁, in human sera.
External Organisation(s): University of Münster
Type: Article
Journal: Analytical chemistry
Volume: 66
Pages: 4027-4033
No. of pages: 7
ISSN: 0003-2700
Publication date: 15.11.1994
Publication status: Published
Peer reviewed: Yes
ASJC Scopus subject areas: Analytical Chemistry
Electronic version(s): https://doi.org/10.1021/ac00094a027 (Access: Unknown)

BibTeX

@article{c30db0f029c64132a0f91a50d80f033f,
title = "Fluorescein Isothiocyanate-Labeled Protein G as an Affinity Ligand in Affinity/Immunocapillary Electrophoresis with Fluorescence Detection",
abstract = "Antibodies from human sera (h-IgG) were tagged with a fluorescent dye (fluorescein isothiocyanate, FITC) through the affinity reaction of FITC-labeled protein G with the Fc fragment of the antibodies. The complexes were quantified by capillary zone electrophoresis (CZE) within 1 min, i.e., fast enough to prevent their dissociation during the measurement. Conditions for the affinity reaction and the CZE analysis could thus be optimized independently. When an FITC-labeled protein G concentration of 10−6 mol/L was used, h-IgG concentrations between 10−6 and 10−9 mol/L were reproducibly quantified (STD < 2%), using an LIF detector. A correlation coefficient, r2, of 0.9988 was established between the peak height and the IgG concentration. Alternatively, h–IgG containing serum samples and the FITC-labeled protein G were simply injected into the CE capillary in consecutive zones, followed by the application of the electrical field. Within 2 min, the affinity complexes were resolved and the IgG content of the serum quantified (r2 = 0.9986). The injection sequence was of no consequence. The measurements agreed well with those found in a single radial immunodiffusion (SRID) assay. In addition FITC-labeled protein G-tagged anti-h-IgG1 antibodies were used to detect the specific antigen of the involved antibody, namely, h-IgG1, in human sera.",
author = "Relf, {Oscar Werner} and Ruth Freitag and Ralf Lausch and Thomas Scheper",
year = "1994",
month = nov,
day = "15",
doi = "10.1021/ac00094a027",
language = "English",
volume = "66",
pages = "4027--4033",
journal = "Analytical chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "22",
}