Development of a chip-based capillary gel electrophoresis method for quantification of a half-antibody in immunoglobulin G4 samples.
A method based on microfluidic technology was developed to support quantitative analysis of recombinant monoclonal immunoglobulin G4 (IgG4) antibody samples. The assay was performed on an Agilent 2100 Bioanalyzer in combination with the Protein 200 Plus LabChip Kit and the Protein 200 Plus assay software. Capillary electrophoresis principles have been transferred to a chip format that integrates all separation, staining, virtual destaining, and detection steps. The method is referred to in this paper as chip-based capillary gel electrophoresis (GelChip-CE method). The GelChip-CE method under nonreducing conditions proved to be a quantitative test for half-antibody determination in IgG4 samples. Similar to the traditional nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method, the GelChip-CE method includes a denaturing step prior to separation. We showed that denaturing the sample by heating resulted in an artificial increase in the amount of half-antibody detected, which could be prevented by addition of N-ethylmaleimide to the sample buffer. The GelChip-CE method allowed for analysis of IgG4 samples with more accuracy, higher precision, and a faster turnaround time than SDS-PAGE and reversed-phase high-performance liquid chromatography (RP-HPLC).
Vasilyeva
Elena
E
BiogenIdec, Fourteen Cambridge Center, Cambridge, MA 02142, USA. elena.vasilyeva@biogenidec.com
Woodard
James
J
Taylor
Frederick R
FR
Kretschmer
Matthias
M
Fajardo
Hans
H
Lyubarskaya
Yelena
Y
Kobayashi
Kazumi
K
Dingley
Amy
A
Mhatre
Rohin
R
D000911:Antibodies, Monoclonal / Q000032:analysis*; D046209:Electrophoresis, Microchip / Q000379:methods* / Q000592:standards; D005033:Ethylmaleimide; D007074:Immunoglobulin G / Q000032:analysis*; D011489:Protein Denaturation; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins / Q000032:analysis; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; D005033:Ethylmaleimide