IMMUNOGLOBULIN CLONALITY ANALYSIS - RESOLUTION OF AMBIGUITIES IN IMMUNOFIXATION ELECTROPHORESIS RESULTS BY HIGH-RESOLUTION, 2-DIMENSIONAL ELECTROPHORETIC ANALYSIS OF PARAPROTEIN BANDS ELUTED FROM AGAROSE GELS

This article describes evaluation of the clonality of origin of immuno globulin (Ig) chains by strategies that include qualitative analysis o f paraproteins with high-resolution, two-dimensional electrophoresis ( 2DE) and silver staining. This approach is helpful in evaluating speci mens in which standard immunoelectrophoresis or immunofixation electro phoresis (IFE) techniques do not provide definitive Ig typing results. One method the authors developed involves a ''band elution'' procedur e, in which proteins present in high-resolution agarose gel electropho resis bands are cut from the agarose gel, eluted with a denaturing buf fer, and subjected to 2DE. The microheterogeneity patterns of the Ig l ight chains, heavy chains, or both are evaluated for their relationshi p regarding mass, charge, and, by inference, number of genes of origin . When necessary, determination of charge relationships may be aided b y urea-mediated carbamylation of lysine residues, which introduces sin gle-charge shifts to the individual protein subunits. Overall, these a djunctive techniques are particularly useful in cases with multiple ba nds of identical immunologic types (eg, several IgG1 bands) on IFE bef ore and after sulfhydryl reduction (with dithiothreitol or 2-mercaptoe thanol). The authors present procedural details and examples of the 2D E band elution patterns of serum and urine samples from four patients with B-cell dyscrasias, including the first reported case of POEMS syn drome with biclonal gammopathy, to the best of their knowledge.