Successful Urine Multiplex Bead Assay to Measure Lupus Nephritis Activity

Abstract

Introduction: Lupus nephritis (LN) confers a poor prognosis, mainly from lack of effective laboratory tests to diagnose and to evaluate therapies. We have previously shown that a set of 6 urinary biomarkers (NGAL, KIM-1, MCP-1, adiponectin, hemopexin, and ceruloplasmin) are highly sensitive and specific to identify adult and pediatric patients with active LN using renal biopsy as reference standard. Using these combinatorial urinary biomarkers, the Renal Activity Score for Lupus (RAIL) score was established, with biomarkers measured by enzyme-linked immunosorbent assay (ELISA). To enhance clinical utility of the biomarkers and RAIL, we tested the performance of RAIL with biomarkers measured by ELISA to that of biomarkers measured by the bead multiplex method, hypothesizing that the multiplex bead method would be comparable.

Methods: Spot urine samples (n = 341) of 46 patients aged 20 to 73 years with or without LN were used. Samples were assayed both by ELISA and multiplex using LUMINEX. RAIL scores and biomarker quantities were assessed for agreement with intraassay correlation coefficients and compared using Bland-Altman and regression.

Results: Biomarker measurement by LUMINEX was successful for NGAL, KIM-1, MCP-1, and adiponectin, but not for ceruloplasmin and hemopexin. There was good agreement of the RAIL obtained from these 4 biomarkers, irrespective of assay method (intraclass correlation coefficient [ICC] = 0.78, 95% confidence interval [CI] = 0.78-0.82). The RAIL scores from 4 biomarkers further correlated with those when considering all 6 biomarkers (ICC = 0.97, 95% CI = 0.96-0.98).

Conclusion: The LUMINEX platform allows for the convenient and simultaneous measurement of 4 RAIL biomarkers. RAIL scores considering only these 4 biomarkers may be sufficient to accurately capture LN activity.

Keywords: ELISA; RAIL; SLE; lupus; multiplex; nephritis.

Graphical abstract

Figure 1

(a) Neutrophil gelatinase−associated lipocalin (NGAL) scatter plot. Comparison of enzyme-linked immunosorbent assay (ELISA) and multiplex for NGAL, with R value of 0.78. (b) NGAL Bland−Altman plot. Mean difference of 0.669, denoted by central line (95% confidence interval = 0.57−0.77). In all, 96% of differences will be between −1.17 and 2.50 (the 95% limit of agreement denoted by lines).

Figure 2

(a) Kidney injury molecule−1 (KIM-1) scatter plot. Comparison of enzyme-linked immunosorbent assay (ELISA) and multiplex for KIM-1, with R value of 0.87. (b) KIM-1 Bland−Altman plot. Mean difference of 0.047, denoted by central line with 95% confidence interval −0.03 to 0.12. In all, 97% of differences will be between −1.28 and 1.38 (the 95% limit of agreement denoted dashed lines).

Figure 3

(a) Monocyte chemoattractant protein−1 (MCP-1) scatter plot. Comparison of enzyme-linked immunosorbent assay (ELISA) and multiplex for MCP-1, with R value of 0.93. (b) MCP-1 Bland−Altman plot. Mean difference of 0.046, denoted by the central line, with 95% confidence interval of −0.01 to 0.1. In all, 96% of mean differences will be between −0.94 and 1.03 (the 95% limit of agreement denoted by dashed lines).

Figure 4

(a) Adiponectin scatter plot. Comparison of enzyme-linked immunosorbent assay (ELISA) and multiplex for adiponectin, with R value of 0.75. (b) Adiponectin Bland−Altman plot. Mean difference of −0.87, denoted by middle line, with 95% confidence interval of −0.97 to −0.77. In all, 96% of mean differences will be between −2.59 and 0.85 (the 95% limits of agreement denoted by dashed lines).

Figure 5

(a) Modified Renal Activity Score for Lupus (RAIL) score scatter plot. Comparison of enzyme-linked immunosorbent assay (ELISA) and multiplex for RAIL score calculated with 4 urinary biomarkers (excludes hemopexin and ceruloplasmin), with R value of 0.65. (b) Modified RAIL score Bland−Altman Plot. Bland−Altman Plot for RAIL score calculated with 4 urinary biomarkers. Mean difference of −1.14, denoted by dashed lines, with 95% confidence interval of −1.24 to 1.03. In all, 95% of values fall between −2.86 and 0.76 (the 95% limits of agreement denoted by the dashed lines).

Figure 6

(a) Scatter plot comparison of modified versus original Renal Activity Score for Lupus (RAIL) score. RAIL scores calculated with 4 (excludes hemopexin and ceruloplasmin) versus 6 urinary biomarkers via enzyme-linked immunosorbent assay (ELISA) methodology, with R value of 0.97. (b) ELISA Bland−Altman Plot. Comparison of modified versus original RAIL score. RAIL scores calculated with 4 (excludes hemopexin and ceruloplasmin) versus 6 urinary biomarkers via ELISA methodology. Mean difference of 1.98, denoted by dashed line (95% confidence interval = 1.95−2.2). In all, 94% of the values fell between 1.37 and 2.59 (the 95% limits of agreement denoted by the dashed lines).