Fluorescein isothiocyanate-labeled protein G as an affinity ligand in affinity/immunocapillary electrophoresis with fluorescence detection.
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.
Reif
O W
OW
Institüt für Technische Chemie, Universität Hannover, Germany.
Lausch
R
R
Scheper
T
T
Freitag
R
R
D004586:Electrophoresis; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D007074:Immunoglobulin G / Q000097:blood* / Q000378:metabolism; D008024:Ligands; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D015203:Reproducibility of Results
C029152:G-substrate; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D008024:Ligands; D009419:Nerve Tissue Proteins; D016650:Fluorescein-5-isothiocyanate