Bender Diane Elizabeth, Schaettler Maximilian O, Sheehan Kathleen Cf, Johanns Tanner M, Dunn Gavin P
The Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St. Louis, MO, USA.
Department of Neurological Surgery, Washington University School of Medicine, St. Louis, MO, USA.
Biomark Insights. 2021 Mar 30;16:11772719211006666. doi: 10.1177/11772719211006666. eCollection 2021.
We compared the performance of two 96-well multiplex immunoassay platforms in assessing plasma cytokine concentrations in patients with glioblastoma (GBM; n = 27), individuals with melanoma, breast or lung cancer metastases to the brain (n = 17), and healthy volunteers (n = 11). Assays included a bead-based fluorescence MILLIPLEX assay/Luminex (LMX) platform and 4 planar electrochemiluminescence kits from Meso Scale Discovery (MSD). The LMX kit evaluated 21 cytokines and the 3 MSD kits evaluated 20 cytokines in total, with 19 overlapping human cytokines between platforms (GM-CSF, IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-21, IL-23, MIP-1α, MIP-1β, MIP-3α, TNFα). The MSD platform had lower LLoQs (lower limits of quantification) than LMX for 17/19 cytokines, and higher LLoQs for IFN-γ and IL-21. The ULoQs were higher in LMX versus MSD assays for 17/19 shared analytes, but lower than MSD for IL-17A and IL-21. With LMX, all 19 shared analytes were quantifiable in each of 55 samples. Although MSD recombinant protein standard curves indicated lower LLoQs than LMX for most cytokines, MSD detected 7/19 (37%) native analytes in <75% of samples, including 0% detection for IL-21 and 8% for IL-23. The LMX platform categorized identical samples at greater concentrations than the MSD system for most analytes (MIP-1β the sole exception), sometimes by orders of magnitude. This mismatched quantification paradigm was supported by Bland-Altman analysis. LMX identified significantly elevated levels of 10 of 19 circulating cytokines in GBM: GM-CSF, IFN-γ, IL-1β, IL-5, IL-10, IL-17A, IL-21, IL-23, MIP-1α, and MIP-3α, consistent with prior findings and confirming the utility of applying appropriate multiplex immunoassay technologies toward developing a cytokine signature profile for GBM.
我们比较了两种96孔多重免疫分析平台在评估胶质母细胞瘤（GBM；n = 27）患者、脑转移黑色素瘤、乳腺癌或肺癌患者（n = 17）以及健康志愿者（n = 11）血浆细胞因子浓度方面的性能。检测方法包括基于微珠的荧光MILLIPLEX检测/Luminex（LMX）平台和来自Meso Scale Discovery（MSD）的4种平面电化学发光试剂盒。LMX试剂盒评估了21种细胞因子，3种MSD试剂盒共评估了20种细胞因子，两个平台之间有19种重叠的人类细胞因子（GM-CSF、IFNγ、IL-1β、IL-2、IL-4、IL-5、IL-6、IL-7、IL-8、IL-10、IL-12p70、IL-13、IL-17A、IL-21、IL-23、MIP-1α、MIP-1β、MIP-3α、TNFα）。对于19种细胞因子中的17种，MSD平台的最低定量限（LLoQ）低于LMX，而IFN-γ和IL-21的LLoQ更高。对于19种共享分析物中的17种，LMX检测的最高定量限（ULoQ）高于MSD检测，但IL-17A和IL-21的ULoQ低于MSD。使用LMX时，55个样本中的每一个样本中的所有19种共享分析物均可定量。尽管MSD重组蛋白标准曲线表明大多数细胞因子的LLoQ低于LMX，但MSD在<75%的样本中检测到19种天然分析物中的7种（37%），包括IL-21的检测率为0%，IL-23的检测率为8%。对于大多数分析物（唯一例外是MIP-1β），LMX平台将相同样本分类的浓度高于MSD系统，有时相差几个数量级。这种不匹配的定量模式得到了Bland-Altman分析的支持。LMX在GBM中鉴定出19种循环细胞因子中的10种水平显著升高：GM-CSF、IFN-γ、IL-1β、IL-5、IL-10、IL-17A、IL-21、IL-23、MIP-1α和MIP-3α，这与先前的发现一致，并证实了应用适当的多重免疫分析技术来开发GBM细胞因子特征谱的实用性。
