MonoclonalImmunoglobulins:AffinityBlottingfor Low Concentrationsin Serum

I describe a simple, economical technique for identifying low concentrations of monoclonal immunoglobulins in the pres-ence of excessive amounts of immunoglobulins of other classes. The technique involves binding of specific antibody to nitrocellulose, separating proteins by isoelectric focusing or zone electrophoresis in agarose gels, using capillary transfer to bind proteins to the nitrocellulose via their anti-body affinity, and then detecting transferred proteins with enzyme-labeled antibody. A monoclonal immunoglobulin can be completely characterized in 2 h. No expensive equipment is required. Affinity blotting is about 10-fold as sensitive as native blotting, 100-fold as sensitive as silver staining. Immune fixation is generally considered more sensitive than immunoelectrophoresis for detecting the presence of monoclonal gammopathies. However, the identification of apparent monoclonal immunoglobulins (mlg’s) can still be difficult when the concentration of the mlg is much lower than the concentration of the residual Ig’s, because the reaction of the light chain of the mig is obscured by that of the residual Ig’s. ’ Use of native blotting (1), although far more sensitive than immune fixation, does not overcome the problem, because all Ig’s are nonselectively transferred to the membrane. The light-chain types of low-concentration mlg’s can be determined by using affinity blotting (2, 3). The protein in question is bound to a solid-phase support via an affinity ligand. The transferred protein(s) is then detected with specific probes. For mlg analyses, specific antibody directed against appropriate Ig heavy-chain determinants is used as the ligand, bound to a nitrocellulose membrane (NC). Pro-teins are separated by isoelectric focusing (IEF) or zone electrophoresis (EP) in ultra-thin agarose gels. Proteins capable of binding to the antibody are selectively traris-ferred to the NC by capillary blotting and detected with enzyme-labeled specific antibodies. After presenting prelim-inary data (4), I modified the procedure, simpli1ring it to suit a routine clinical laboratory