Multiplexed Quantification of Human IgG, IgA, and IgM with the FlowMetrixTM System,

A quantitative assay for the three major serum immunoglobulins (Igs) has been developed for the FlowMetrixTM system that performs each measurement simultaneously in the same test tube. The FlowMetrix system is a computer-enhanced flow cytometer that utilizes multiple fluorescent color sets of microspheres to provide a multiplexed format (1)(2). The microspheres are 5.5 μm in diameter and are composed of polystyrene and methacrylate to provide a surface carboxylate functionality. They are dyed internally with a mixture of two fluorophores that enables their classification into 64 distinct color sets. Igs are found in serum at high concentrations, allowing their assay in the competitive inhibition format. Each assay was developed by the attachment of purified IgG, IgA, or IgM to three of the available 64 uniquely fluorescing microsphere sets. Antibody specifically reactive to each Ig class was then labeled with a third fluorophore that acted as the reporter molecule. The amount of this reporter detected on the microsphere surface was inversely proportional to the concentration of soluble Ig in the serum sample. Each assay was conveniently optimized by altering the concentration of reporter antibody to provide a dynamic range encompassing the physiological serum concentrations of each Ig. The significance of the assay was the multiplexed format. IgG, IgA, IgM, and bovine serum albumin (BSA), as a control, were conjugated separately to four distinct microsphere sets with a two-step 1-ethyl-3-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) coupling method. Activated microspheres were suspended in a 0.05 g/L solution of protein in PBS, pH 7.4, for 1 h and then washed twice with 0.2 mL/L Tween 20 and 1 g/L BSA in PBS, pH 7.4 (PBSTB), to block remaining sites. Three affinity-purified goat antibodies to human gamma, alpha, and mu chains were labeled with Bodipy® FL-CASE (Molecular Probes). Antigens and antibodies were obtained from the Cappel Division of Organon Teknika. For the multiplexed assay, equivalent amounts of the four protein-loaded microspheres were mixed. Ten microliters of this blend containing 7500 of each microsphere set was mixed with 10 μL of serum calibrant or serum sample diluted 1:500 in PBSTB. Immediately, 10 μL of a cocktail of the three Bodipy-labeled reporter antibodies at 30, 8, and 2.5 mg/L for anti-gamma, anti-alpha, and anti-mu, respectively, was added and mixed well. After 30 min the mixtures were diluted to 300 μL in PBSTB and assayed with the FlowMetrix system. The FlowMetrix system consists of a Becton Dickinson FACScan flow cytometer interfaced with a personal computer configured with proprietary hardware and software that controls all functions of the cytometer as well as providing real-time data acquisition and analysis for the multiplexed assays. During the 10–15 s of machine time the system first categorized each microsphere according to its fluorescent color set. The system then determined the average green fluorescence for 100 of each color set of microspheres, providing a quantification of that particular analyte. Calibration curves for the multiplexed assay are shown in Fig. 1 . Serum calibrants with known concentrations of IgG, IgA, and IgM were obtained from Kamiya Biomedical Co. These were traceable to the CAP/BCR/IFCC Reference Preparation for Proteins in Human Serum (RPPHS), lot 91/0619. For each curve, a polynomial trendline was used for nonlinear regression analysis with calibrators at 3–30 g/L for IgG, 0.6–5.2 g/L for IgA, and 0.36–3.0 g/L for IgM to cover the concentration range of that Ig in serum (3). Three seven-point calibration curves were generated in only seven test tubes. Imprecision of the multiplexed assay was determined on three consecutive days by assaying low- (Level 1) and high- (Level 2) concentration controls (LiquichekTM, Bio-Rad) with values traceable to IFCC reference preparations. Controls were run in replicates of five, and imprecision was reported as the within-run and between-run CVs (Table 1 ). The within-run CVs for the three assays ranged from 2.9% for the high control of IgA to 6.2% for the high control of IgG. Five of the six between-run CVs were <6.5%. The low IgM assay provided a less precise interassay CV of 15%. Although technically not measuring accuracy, the mean concentrations of the controls for the IgG, IgA, and IgM determinations (n = 15 for each control) were all within the acceptable range according to the package insert of the Liquichek calibrators. Multiplexed FlowMetrix analysis was compared with rate immunonephelometry by using 50 serum samples provided by Laboratory Corp. of America. Samples were assayed with the Beckman Array® rate immunonephelometry system and then transported to Luminex Corp. for FlowMetrix analysis. Results were compared by linear least-squares regression analysis (Table 1 ). The IgA and IgM results for FlowMetrix correlated well with the nephelometric results. The IgG results demonstrated a high intercept with a slope of 0.74. The primary reason for these results was the design of the assay. All three of these assays were designed to measure concentrations in typical adult sera. Low-end sensitivity was deemed less important, resulting in a limit of detection (LOD) for IgG that is quite high at 3.0 g/L. One would thus expect correlation with assays that possess lower LOD, such as nephelometry, to produce high intercepts. As the multiplexed FlowMetrix assay was performed on serum diluted 1:500, use of a less-dilute sample may improve the agreement with the nephelometric method. We conclude that this multiplexed, competitive inhibition assay for human serum IgG, IgA, and IgM demonstrated acceptable precision and agreement with an immunonephelometic method. Luminex Corp., 12212 Technology Blvd., Austin, TX 78727 Calibration curves for the three multiplexed assays. Results of a seven-point calibration curve with calibrants of known IgG (♦), IgA (•), and IgM (▪) concentrations. As the concentration of serum Ig increases, there is a corresponding decrease in the mean fluorescence intensity (green). Imprecision of the FlowMetrix multiplexed assay and correlation with the Beckman Array®. g/L. n = 15. n = 3. Imprecision of the FlowMetrix multiplexed assay and correlation with the Beckman Array®. g/L. n = 15. n = 3.