An ellipsoidal mirror for detection of laser-induced fluorescence in capillary electrophoresis system: applications for labelled antibody analysis.
An LIF detector was integrated into a CE system which uses a ball lens to focus the laser beam on the CE capillary. The detector employs an ellipsoid that is glued on the capillary window, to permit the collection of the fluorescence in the capillary. This 'trapped' fluorescence stays in the capillary because the angle of the silica/air interface is greater than the critical angle. The performance of this new detector setup is found to be identical to the collinear setup using the same ball lens. An application to the analysis of FITC-labeled IgG was optimized using a 14 cm effective length capillary. The LOD of an FITC-labeled IgG2 at an excitation wavelength of 488 nm was 150 pg/mL, which was 10 times better than the LOD recorded with slab gel silver staining. Using a tetramethylrhodamine (TAMRA)-labeled IgG2 and a 532 nm excitation wavelength the LOD is 50 pg/mL. The electropherograms of four different commercial FITC conjugates of IgG were studied. The presence of aggregates was observed in two samples while close kinetics of reduction was observed between free aggregates and high aggregates concentration samples. The integrated LIF detector provides an extremely powerful and convenient tool for antibody analysis and should be useful for therapeutic MAb control in pharmaceutical facilities.
Rodat
Audrey
A
Laboratoire des IMRCP, Université de Toulouse, Université Paul Sabatier, Toulouse, France.
Kalck
Fabien
F
Poinsot
Véréna
V
Feurer
Bernard
B
Couderc
François
F
D000818:Animals; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification; D007834:Lasers; D051379:Mice; D011817:Rabbits
D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D016650:Fluorescein-5-isothiocyanate