A method for recovery of native, clonally-restricted immunoglobulins from agarose gels.
Multiple low level, clonally-restricted, immunoglobulins (Ig) are commonly encountered on routine serum protein electrophoresis by clinical laboratories using high resolution zone electrophoresis on agarose. We sought a method for recovering the clonally-restricted Ig, in native configuration, from clinical laboratory gels as a first step in the investigation of its clinical significance. We found that a two-stage electrophoretic procedure gave consistently good recoveries. After routine agarose gel electrophoresis, portions of the electropherogram, containing clonally-restricted Ig, were excised and subjected to flatbed isoelectric focusing in agarose to enhance separation of the individual antibody clonotypes. Multiple slabs, containing the same clonally-restricted Ig, could be cut from adjacent tracks (i.e., tracks loaded with the same specimen) on the zone electropherogram and applied to a single track on the focusing gel to improve separation and increase yields. The focused gels were cut to isolate slabs containing individual clonotypes. These slabs were washed to remove carrier ampholytes and held at -20 degrees C overnight. Ig was extracted from the thawed gels, with 61-68% recovery, by ultracentrifugation following physical disruption of the gel. Antigen binding activity of the recovered Ig was verified by rate nephelometry. Clonally-restricted antibodies were successfully isolated from an immune animal serum by this procedure and biotinylated for use as probes on Western blots.
Janson
R W
RW
Departments of Medicine, University of Pittsburgh School of Medicine, PA.
Vertosick
F T
FT
Kelly
R H
RH
D004586:Electrophoresis / Q000379:methods*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007136:Immunoglobulins / Q000302:isolation & purification
D007074:Immunoglobulin G; D007136:Immunoglobulins