Bastarache J A, Koyama T, Wickersham N E, Ware L B
Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University School of Medicine, T-1218 MCN, Nashville, TN 37232-2650, United States.
Department of Biostatistics, Vanderbilt University School of Medicine, T-1218 MCN, Nashville, TN 37232-2650, United States.
J Immunol Methods. 2014 Jun;408:13-23. doi: 10.1016/j.jim.2014.04.006. Epub 2014 Apr 21.
Despite the widespread use of multiplex immunoassays, there are very few scientific reports that test the accuracy and reliability of a platform prior to publication of experimental data. Our laboratory has previously demonstrated the need for new assay platform validation prior to use of biologic samples from large studies in order to optimize sample handling and assay performance.
In this study, our goal was to test the accuracy and reproducibility of an electrochemiluminescent multiplex immunoassay platform (Meso Scale Discovery, MSD®) and compare this platform to validated, singleplex immunoassays (R&D Systems®) using actual study subject (human plasma and mouse bronchoalveolar lavage fluid (BAL) and plasma) samples.
We found that the MSD platform performed well on intra- and inter-assay comparisons, spike and recovery and cross-platform comparisons. The mean intra-assay CV% and range for MSD were 3.49 (0.0-10.4) for IL-6 and 2.04 (0.1-7.9) for IL-8. The correlation between values for identical samples measured on both MSD and R&D was R=0.97 for both analytes. The mouse MSD assay had a broader range of CV% with means ranging from 9.5 to 28.5 depending on the analyte. The range of mean CV% was similar for single plex ELISAs at 4.3-23.7 depending on the analyte. Regardless of species or sample type, CV% was more variable at lower protein concentrations.
In conclusion, we validated a multiplex electrochemiluminescent assay system and found that it has superior test characteristics in human plasma compared to mouse BALF and plasma. Both human and MSD assays compared favorably to well-validated singleplex ELISAs.
尽管多重免疫测定已广泛应用，但在发表实验数据之前，很少有科学报告对平台的准确性和可靠性进行测试。我们实验室之前已证明，在使用来自大型研究的生物样本之前，需要对新的检测平台进行验证，以优化样本处理和检测性能。
在本研究中，我们的目标是测试电化学发光多重免疫测定平台（Meso Scale Discovery，MSD®）的准确性和可重复性，并使用实际研究对象（人血浆、小鼠支气管肺泡灌洗液（BAL）和血浆）样本将该平台与经过验证的单重免疫测定（R&D Systems®）进行比较。
我们发现MSD平台在批内和批间比较、加标回收和跨平台比较中表现良好。MSD检测IL-6的批内CV%均值及范围为3.49（0.0 - 10.4），检测IL-8的批内CV%均值及范围为2.04（0.1 - 7.9）。在MSD和R&D上测量的相同样本的值之间的相关性，两种分析物的R均为0.97。小鼠MSD检测的CV%范围更广，均值根据分析物在9.5至28.5之间。单重ELISA的平均CV%范围类似，根据分析物在4.3 - 23.7之间。无论物种或样本类型如何，在较低蛋白质浓度下CV%的变化更大。
总之，我们验证了一种多重电化学发光检测系统，发现它在人血浆中的检测特性优于小鼠BALF和血浆。人与MSD检测与经过充分验证的单重ELISA相比均表现良好。
