Cody Ellen M, Sproles Alyssa, Rose James, Huang Bin, Devarajan Prasad, Brunner Hermine I, Thornton Sherry
Division of Pediatric Nephrology, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Division of Rheumatology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.
Kidney Int Rep. 2025 Apr 21;10(7):2255-2264. doi: 10.1016/j.ekir.2025.04.013. eCollection 2025 Jul.
We aimed to develop multiplex (MLP) assays of 6 biomarkers, namely adiponectin, neutrophil gelatinase-associated lipocalin (NGAL), monocyte chemoattractant protein-1 (MCP-1), kidney injury molecule-1 (KIM-1), ceruloplasmin, and hemopexin used in the Renal Activity Index for Lupus (RAIL) and establish MLP assays using the Milliplex MLP and the electrochemiluminescence Mesoscale Discovery (MSD) technology, to compare with the gold standard of established single immunoassays.
A total of 104 banked urine samples from the CCHMC Lupus Cohort were used. RAIL biomarker concentrations were assayed using established individual immunoassays, and concentrations were compared with MLP reagents using both the MSD and MLP platforms. MLP assay development involved assessment of biomarker concentrations in 40 individual urine samples, followed by evaluation of optimal sample dilution using 14 additional samples on each platform. Then 50 samples were assayed in duplicate under optimized MLP conditions, and biomarker concentration compared with those using single assays. After correcting for urine creatinine, RAIL scores of the samples were determined and compared between testing platforms (single immunoassays, MLP).
Our results indicate that a 1:25 urine dilution was optimal when using the MLP platforms. Biomarker concentrations by single immunoassays correlated with those on the Milliplex platform strongly for KIM-1, MCP-1, and NGAL ( = 0.726-0.86, < 0.0001), moderately for adiponectin ( = 0.629, < 0.0001) and weakly for ceruloplasmin ( = 0.367, = 0.009). Using the MSD platform, comparatively lower correlations with those by single immunoassay were observed (NGAL: = 0.516, = 0.0001; adiponectin and hemopexin: ≤ 0.29, = 0.042; ceruloplasmin, KIM-1, and MCP-1: all < 0.2).
Milliplex technology is suitable to measure RAIL biomarker concentrations in urine samples diluted 1:25.
我们旨在开发一种包含6种生物标志物的多重检测（MLP）方法，这6种生物标志物分别是脂联素、中性粒细胞明胶酶相关脂质运载蛋白（NGAL）、单核细胞趋化蛋白-1（MCP-1）、肾损伤分子-1（KIM-1）、铜蓝蛋白和血红素结合蛋白，这些标志物用于狼疮肾活性指数（RAIL），并使用Milliplex MLP和电化学发光Mesoscale Discovery（MSD）技术建立MLP检测方法，以与已确立的单一免疫检测的金标准进行比较。
使用了来自辛辛那提儿童医院医疗中心狼疮队列的104份储存尿液样本。使用已确立的个体免疫检测方法测定RAIL生物标志物浓度，并使用MSD和MLP平台将浓度与MLP试剂进行比较。MLP检测方法的开发包括评估40份个体尿液样本中的生物标志物浓度，随后在每个平台上使用另外14份样本评估最佳样本稀释度。然后在优化的MLP条件下对50份样本进行重复检测，并将生物标志物浓度与使用单一检测方法的结果进行比较。在校正尿肌酐后，确定样本的RAIL评分并在不同检测平台（单一免疫检测、MLP）之间进行比较。
我们的结果表明，使用MLP平台时，1:25的尿液稀释度是最佳的。单一免疫检测的生物标志物浓度与Milliplex平台上的浓度在KIM-1、MCP-1和NGAL方面相关性很强（r = 0.726 - 0.86，P < 0.0001），在脂联素方面相关性中等（r = 0.629，P < 0.0001），在铜蓝蛋白方面相关性较弱（r = 0.367，P = 0.009）。使用MSD平台时，观察到与单一免疫检测结果的相关性相对较低（NGAL：r = 0.516，P = 0.0001；脂联素和血红素结合蛋白：r ≤ 0.29，P = 0.042；铜蓝蛋白、KIM-1和MCP-1：均r < 0.2）。
Milliplex技术适用于测量稀释度为1:25的尿液样本中的RAIL生物标志物浓度。
