IgG heavy-chain sublcass typing of myeloma paraproteins by isoelectric focusing immunoblot analysis

The objective of this study was to develop a clinical laboratory method for subclass typing of human immunoglobulin G (IgG) paraproteins. Serum proteins were isoelectrically focused (IEF) in a minigel and passively blotted by capillary diffusion onto untreated nitrocellulose. Unreacted sites on the nitrocellulose were blocked with bovine serum albumin and the bound IgG was detected with peroxidase-conjugated anti-human IgG_1-4 monoclonal antibodies from WHO/IUIS clones. The IEF immunoblot specificity was demonstrated by analysis of documented IgG, IgA, and IgM myeloma proteins of known subclass and light-chain composition. IEF immunoblots of sera from 18 myeloma patients who had an above-normal total IgG concentration produced IEF immunoblot patterns composed of five to 10 discrete bands (pI range 6.0 to 8.4). In contrast, no detectable IgG bands were observed with sera containing IgA and IgM paraproteins. The observed subclass frequencies of IgG paraproteins were 56% IgG₁ (10/18), 28% IgG₂ (5/18), 11% IgG₃ (2/18), and 5% IgG₄ (1/18). IEF immunoblot analysis permits the monitoring of changes in the pI and subclass of an IgG paraprotein over the course of a myeloma patient's therapy program.