Analysis of immunoglobulin G using a capillary electrophoretic affinity assay with protein A and laser-induced fluorescence detection.
A method for the rapid and sensitive determination of immunoglobulin G (IgG) in cultivation media by an affinity assay using capillary electrophoresis is presented. For that purpose we evaluated protein A conjugated with a fluorescent dye such as fluorescein diisocyanate or dichlorotriazinyl-aminofluorescein as an affinity ligand. The ligand formed a fluorescing complex with immunoglobulin G in the sample and rapid separation from excess protein A was performed by capillary zone electrophoresis. However, only partial resolution of the zones was achieved when protein A as a whole molecule was utilized. In contrast, baseline resolution of the zones was obtained when recombinant fragments of protein A were used as affinity ligands. Immunoglobulin concentrations in the range of two orders of magnitude were determined. Due to the specificity of protein A for immunoglobulin G, analysis can be carried out even in the presence of high concentrations of other components and in cultivation media. Thus, the capillary electrophoretic affinity assay was successfully applied to monitor monoclonal antibodies in a cultivation process.
Lausch
R
R
Institut für Biochemie, Abteilung Biotechnologie, Westfälische Wilhelms-Universität Münster, Germany.
Reif
O W
OW
Riechel
P
P
Scheper
T
T
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D003470:Culture Media / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D007122:Immunoelectrophoresis / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis*; D007834:Lasers; D008024:Ligands; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D003470:Culture Media; D005452:Fluoresceins; D007074:Immunoglobulin G; D008024:Ligands; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; C039083:5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)fluorescein; D016650:Fluorescein-5-isothiocyanate