title: Development of a highly sensitive three-dimensional gel electrophoresis method for characterization of monoclonal protein heterogeneity.
abstract: Three-dimensional gel electrophoresis (3-DE), which combines agarose gel electrophoresis and isoelectric focusing/SDS-PAGE, was developed to characterize monoclonal proteins (M-proteins). However, the original 3-DE method has not been optimized and its specificity has not been demonstrated. The main goal of this study was to optimize the 3-DE procedure and then compare it with 2-DE. We developed a highly sensitive 3-DE method in which M-proteins are extracted from a first-dimension agarose gel, by diffusing into 150 mM NaCl, and the recovery of M-proteins was 90.6%. To validate the utility of the highly sensitive 3-DE, we compared it with the original 3-DE method. We found that highly sensitive 3-DE provided for greater M-protein recovery and was more effective in terms of detecting spots on SDS-PAGE gels than the original 3-DE. Moreover, highly sensitive 3-DE separates residual normal IgG from M-proteins, which could not be done by 2-DE. Applying the highly sensitive 3-DE to clinical samples, we found that the characteristics of M-proteins vary tremendously between individuals. We believe that our highly sensitive 3-DE method described here will prove useful in further studies of the heterogeneity of M-proteins.
authors.affiliation: Clinical Laboratory, Hokkaido University Hospital, Sapporo, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Nakano
authors.forename: Shogo
authors.initials: S
authors.lastname: Tamura
authors.forename: Kohei
authors.initials: K
authors.lastname: Otuka
authors.forename: Noriyasu
authors.initials: N
authors.lastname: Niizeki
authors.forename: Masahiko
authors.initials: M
authors.lastname: Shigemura
authors.forename: Chikara
authors.initials: C
authors.lastname: Shimizu
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Matsuno
authors.forename: Seiichi
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Takanori
authors.initials: T
authors.lastname: Moriyama
mesh_terms: D000368:Aged; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics / Q000378:metabolism*; D007525:Isoelectric Focusing / Q000379:methods; D008875:Middle Aged; D009101:Multiple Myeloma / Q000378:metabolism*; D012680:Sensitivity and Specificity
chemical_list: D007136:Immunoglobulins