title: Development of a highly-sensitive multi-plex assay using monoclonal antibodies for the simultaneous measurement of kappa and lambda immunoglobulin free light chains in serum and urine.
abstract: Monoclonal κ and λ immunoglobulin free light chain (FLC) paraproteins in serum and urine are important markers in the diagnosis and monitoring of B cell dyscrasias. Current nephelometric and turbidimetric methods that use sheep polyclonal antisera to quantify serum FLC have a number of well-observed limitations. In this report, we describe an improved method using specific mouse anti-human FLC monoclonal antibodies (mAbs). Anti-κ and anti-λ FLC mAbs were, separately, covalently coupled to polystyrene Xmap® beads and assayed, simultaneously, in a multi-plex format by Luminex® (mAb assay). The mAbs displayed no cross-reactivity to bound LC, the alternate LC type, or other human proteins and had improved sensitivity and specificity over immunofixation electrophoresis (IFE) and Freelite™. The assay gives good linearity and sensitivity (<1 mg/L), and the competitive inhibition format gave a broad calibration curve up to 437.5 mg/L and prevented anomalous results for samples in antigen excess i.e. high FLC levels. The mAbs displayed good concordance with Freelite™ for the quantitation of normal polyclonal FLC in plasma from healthy donors (n=249). The mAb assay identified all monoclonal FLC in serum from consecutive patient samples (n=1000; 50.1% with monoclonal paraprotein by serum IFE), and all FLC in a large cohort of urine samples tested for Bence Jones proteins (n=13090; 22.8% with monoclonal κ, 9.0% with monoclonal λ, and 0.8% with poly LC detected by urine IFE). Importantly this shows that the mAbs are at least close to the ideal of detecting FLC from all patients and neoplastic plasma cell clones. Given the overall effectiveness of the anti-FLC mAbs, further clinical validation is now warranted on serial samples from a range of patients with B cell disorders. Use of these mAbs on other assay platforms should also be investigated.
authors: Clinical Immunology Service, School of Immunity and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.
authors: John P
authors: JP
authors: Campbell
authors: Mark
authors: M
authors: Cobbold
authors: Yanyun
authors: Y
authors: Wang
authors: Margaret
authors: M
authors: Goodall
authors: Sarah L
authors: SL
authors: Bonney
authors: Anita
authors: A
authors: Chamba
authors: Jane
authors: J
authors: Birtwistle
authors: Timothy
authors: T
authors: Plant
authors: Zaheer
authors: Z
authors: Afzal
authors: Roy
authors: R
authors: Jefferis
authors: Mark T
authors: MT
authors: Drayson
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001667:Binding, Competitive; D014408:Biomarkers, Tumor / Q000097:blood* / Q000652:urine*; D002138:Calibration; D016022:Case-Control Studies; D003429:Cross Reactions; D006801:Humans; D007118:Immunoassay / Q000592:standards; D007145:Immunoglobulin kappa-Chains / Q000097:blood* / Q000652:urine*; D007146:Immunoglobulin lambda-Chains / Q000097:blood* / Q000652:urine*; D016014:Linear Models; D051379:Mice; D008807:Mice, Inbred BALB C; D009101:Multiple Myeloma / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000652:urine; D011237:Predictive Value of Tests; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D007145:Immunoglobulin kappa-Chains; D007146:Immunoglobulin lambda-Chains