IMMUNOMETRIC ASSAY TO DETERMINE FREE LIGHT CHAIN CONCENTRATIONS OF HUMAN IMMUNOGLOBULINS

Abstract

Detection of total free light chains (FLC) of immunoglobulins and their ratio (kappa/lambda quotient) are used in diagnostics and monitoring of multiple myeloma and other gammapathies, primary amyloidosis and multiple sclerosis. Previously described immunoassays with monoclonal antibodies (Mabs) against cryptic and constantly exposed epitopes of FLC failed to recognize rare variants of lambda Bence-Jones proteins and a significant proportion of lambda chains excreted with urine. Aiming to improve this approach, a novel murine Mab (IgG2b coded as 1C8) was employed, which specifically binds free lambda chains but doesn’t interact with native IgA, IgG, and IgM. The novel Mab recognized an epitope exposed at free lambda chains in peripheral blood of healthy donors and patients with multiple myeloma. It is not destroyed or masked upon renal filtration. The aim of this study was to determine basic features of improved assay system, and to estimate its potential in diagnostics of monoclonal gammapathies. The mixtures of three Bence-Jones proteins of either kappa- or lambda- types purified from the urine of multiple myeloma patients were used as calibrator samples. Improved immunometric assay is able to detect free kappa and lambda chains in serum and urine at a scale of 1 to 100 ng/ml, thus being three orders more sensitive than, e.g., detection levels of Freelite method based on polyclonal antibodies. A novel assay allows to detect free kappa and lambda chains at comparable levels in serum or urine, and to deduce kappa/lambda ratio. The proposed assay is able to detect FLC in 10,000-fold excess of whole IgG molecules. The calibrating plots for both antigens are linear on log-log scales, with very similar slopes. Detection thresholds for kappa or lambda chains proved to be 5 and 3 ng/ml, respectively. Mean concentrations of free kappa chains in sera of healthy donors were 6.7±2.1, in urine, 4.2±3.8 mcg/ml. Mean concentrations of free lambda chains were 4.7±1.96, and 1.6±1.0 mcg/ml, respectively. This method, if applied to serum and urine samples from multiple myeloma patients, revealed free light chains were similar to the paraproteins detected by means of electrophoresis/immunofixation. The values of kappa/lambda ratios corresponded to the types of gammapathies revealed.