These spiked

samples were serially diluted 1 in 4 in assay buffer and measured on the mAb assay. Serial dilutions DNA Damage inhibitor were replicated five times within the same plate, and the limit of detection for each mAb was then assessed. The limit of detection for each mAb was determined by selecting the lowest concentration detected by the mAb assay above the blank well containing only assay buffer (no BJ protein). Assay linearity was assessed by serially diluting three serum samples containing elevated levels of monoclonal κ FLC and three samples containing elevated levels of monoclonal λ FLC, two-fold in assay buffer. These six samples were serially diluted nine times with three replicates of each dilution conducted within the same plate. Linearity of the mAb assay was then assessed on the ten sample dilutions. Because competitive inhibition assays are inherently non-linear, a strategy for demonstrating linearity was conducted by comparing the expected results against the acquired results from the serial dilutions. Assay batch-to-batch variability was assessed by analysing fifty serum samples with varying FLC levels once, on separate assay days, using

three consecutive PF-562271 mouse batches of anti-FLC mAbs, calibrators and other appropriate assay reagents. Assay imprecision was estimated by calculating the intra-assay coefficient of variation percentage (CV%) and the inter-assay CV%. For these tests, pools of samples with low, medium and high levels of κ and λ FLCs were used. All

samples were analysed in duplicate, every morning, for ten working consecutive days, and all tests were conducted in accordance with the Clinical and Laboratory Standards Institute (CLSI) guideline EP5-A2 (Tholen et al., 2004). The susceptibility of the MTMR9 mAb assay to interference was measured by adding known quantities of interference agents to a pool of National Health Service Blood and Transplant Service (NHSBT) plasma samples containing normal κ (11.12 mg/L) and λ (7.62 mg/L) FLC levels. Individual aliquots of the plasma pool were spiked with purified IgG-κ (3.5 g/L), IgG-λ (3.6 g/L), IgA-κ (1.5 g/L), IgA-λ (3.2 g/L), IgM-κ (6.5 g/L), IgM-λ (3.7 g/L), haemoglobin (4 g/L), bilirubin (0.2 g/L), cholesterol (2 g/L), triglyceride (5 g/L), as well as κ FLC (2 g/L) or λ FLC (2 g/L). Interference testing was conducted in accordance with CLSI guidelines EP7-A2 (McEnroe et al., 2005). For all tests on assay dynamics, except mAb limit of detection, the maximal value obtained from each anti-κ FLC mAb (BUCIS 01 or BUCIS 04) was used as the final κ result, and the same approach was used for each anti-λ FLC mAb (BUCIS 03 and BUCIS 09) for λ FLC results. 250 plasma samples obtained from healthy random donors (NHSBT UK) were measured using the Freelite™ and mAb assays; results obtained by all four anti-FLC mAbs were used for these analyses.