Immunoassays for IgG and IgA Light Chain Pairs Are More Sensitive Than Immunofixation Electrophoresis in Multiple Myeloma and Provide Quantitative Analysis

Introduction: Serum free light chain (sFLC) measurements are having a big impact in the diagnosis and monitoring of patients with monoclonal gammopathies. Utility is largely dependent upon assessing FLC kappa/lambda (κ/λ) ratios as the determinant of tumor clonality. To date, intact immunoglobulin measurements comprise total IgG, IgA and IgM, the measurement of bands on gels by serum protein electrophoresis (SPE) or immunofixation electrophoresis (IFE). We hypothesised that analysis of intact immunoglobulin molecules for their light chain content and light chain ratios might be clinically useful in patients with multiple myeloma (MM).

Methods: Monospecific polyclonal antisera were produced against IgGκ, IgGλ, IgAκ and IgAλ, using a combination of tolerisation and affinity chromatography. The four reagents reacted only with conformational epitopes that spanned the quaternary junctional regions between bound κ or λ light chains and their respective heavy chain partners. Antisera were then tailored for use as nephelometric homogeneous immunoassays on a Siemens Dade-Behring BN^™II clinical laboratory analyser. Reagents were assessed for normal ranges (using blood donor sera) and clinical utility in IgG and IgA MM sera.

Results: For IgG, normal median values (and 95% ranges) in 108 sera were: IgGκ 6,981 mg/L (3,608–11,655); IgGλ 3,946 mg/L (2,023–9,158); IgGκ/IgGλ ratio 1.8 (1.15–2.70). Correlation of IgGκ+IgGλ vs total IgG, 0.8: p<0.01 (Pearson’s rank test). For IgA in 191 normal sera, respective values were: IgAκ 1,270 mg/L (440–2,360); IgAλ 870 mg/L (340–1,850); IgAκ/IgAλ ratio 1.47 (0.58–2.52). Correlation of IgAκ+IgAλ vs total IgA, 0.924: p<0.001. For IgG MM sera, sensitivity of the IgGκ/IgGλ ratios were compared to IFE. In all of 19 presentation sera and all samples during serial monitoring of 9 patients (4 IgGκ; 5 IgGλ), the appropriate immunoglobulin results were elevated and IgGκ/IgGλ ratios were abnormal indicating similar or greater sensitivity. In 3/9 patients during monitoring, IgGκ or IgGλ results were abnormal at complete remission (as judged by normal IFE results). Overall, relapse and responses were also detected earlier by IgGκ or IgGλ than by SPE or IFE. In addition, 2/9 patients showed discordant results with no change in IgGκ/IgGλ ratios during chemotherapy but a marked fall in total IgG measurements. For IgA MM presentation sera, all 83 had abnormal IgAκ/IgAλ ratios but 46 could not be quantified by SPE because of diffuse or overlapping protein bands. In 14 patients assessed during disease monitoring, all samples had IgAκ/IgAλ ratios that corresponded with IFE; in 5/14 patients, the results could not be quantified by SPE because IgA bands were masked by other proteins. Kaplan Meier survival curves on patients with ratios (either IgAκ/IgAλ or IgAλκ/IgAκ) greater than 500 (mean 449: range 4.96–3,675) had worse outcome than below 500 ratios; 23 vs 34 months (p<0.05).

Conclusion: Measurements of IgGκ, IgGλ, IgAκ and IgAλ and their ratios were clinically at least as sensitive as SPE and IFE, both at diagnosis and for identifying residual disease. Furthermore, they provided quantitative results compared with the non-quantitative IFE method. Initial results for IgAκ/IgAλ ratios indicate a prognostic value at disease presentation suggesting a better clinical utility than total IgA measurements. Furthermore, IgGκ/IgGλ and IgAκ/IgAλ ratios may provide improved assessment of tumor responsiveness during monitoring in a similar manner to FLC κ/λ ratios. This may be due, in part, to the ratios compensating for changes in blood and plasma volume and reduced expression of FcRn IgG recycling receptors, resulting from chemotherapy.