Young Shih-Houng, Antonini James M, Roberts Jenny R, Erdely Aaron D, Zeidler-Erdely Patti C
Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA.
J Immunol Methods. 2008 Feb 29;331(1-2):59-68. doi: 10.1016/j.jim.2007.11.004. Epub 2007 Dec 17.
There is a growing demand for a cost-effective, efficient, and high-throughput method for measuring cytokines. Currently, many studies are using flow cytometric bead-based multiplex assays in the measurement of cytokines. However, limited data are available regarding the performance of these cytometric bead assays versus enzyme-linked immunosorbent assay (ELISA) or correlation with mRNA expression using real time reverse transcriptase-polymerase chain reaction (RT-PCR). In one of our studies, cytometric bead array (CBA) was used to measure inflammatory cytokine protein levels in bronchoalveolar lavage (BAL) samples from mice exposed to welding fume, an inflammatory particulate. The results were then compared to whole lung mRNA levels of the same cytokines measured by real time RT-PCR in the same mouse model. It was found that the trends in cytokine profiles measured via CBA agreed with the whole lung mRNA results. In a separate experiment, we used a rat zymosan infectivity model to induce a pulmonary immunomodulatory response and determined cytokine concentrations in recovered BAL fluid by ELISA and two different types of cytometric bead-based assays, CBA and FlowCytomix (FC). The sample-to-sample correlation was good between ELISA and CBA with correlation coefficient R values of 0.76, 0.66, and 0.92 for rat IFN-gamma, TNF-alpha, and IL-6, respectively. ELISA only correlated significantly with the FC assay for TNF-alpha with R=0.43. Patterns of cytokine response in our rat model also differed among the assays but overall, the ELISA and CBA yielded similar results. For a method-to-method comparison, we assayed supplied cytokine standards from ELISA kits using both ELISA and CBA to determine the R values and found it to be greater than 0.90 for all the cytokines tested. It was found that the ELISA was more sensitive in the low range of the standard curve while the bead assays were capable of detecting higher protein concentrations, which would allow for direct measurement of concentrated samples. There was a lack of agreement between the absolute protein values for the ELISA and flow cytometric bead-based assays; in most cases, the latter method tended to give higher protein concentrations than ELISA. In conclusion, direct comparisons between absolute protein values did not agree among the assays tested in this study, but patterns of cytokine response generally agreed between ELISA and CBA. In the case of the mouse CBA, a companion measurement is recommended if samples with low concentrations of an analyte are reported and extrapolated below sensitivity or zero.
对于一种经济高效且高通量的细胞因子检测方法的需求日益增长。目前，许多研究在细胞因子检测中使用基于流式细胞术微珠的多重分析方法。然而，关于这些细胞计数微珠分析与酶联免疫吸附测定（ELISA）的性能比较，或与使用实时逆转录聚合酶链反应（RT-PCR）的mRNA表达的相关性，可用数据有限。在我们的一项研究中，细胞计数微珠阵列（CBA）用于测量暴露于焊接烟尘（一种炎性颗粒物）的小鼠支气管肺泡灌洗（BAL）样本中的炎性细胞因子蛋白水平。然后将结果与在同一小鼠模型中通过实时RT-PCR测量的相同细胞因子的全肺mRNA水平进行比较。结果发现，通过CBA测量的细胞因子谱趋势与全肺mRNA结果一致。在另一个实验中，我们使用大鼠酵母聚糖感染模型诱导肺部免疫调节反应，并通过ELISA以及两种不同类型的基于流式细胞术微珠的分析方法（CBA和FlowCytomix，FC）测定回收的BAL液中的细胞因子浓度。ELISA与CBA之间的样本间相关性良好，大鼠干扰素-γ、肿瘤坏死因子-α和白细胞介素-6的相关系数R值分别为0.76、0.66和0.92。ELISA仅与FC分析中肿瘤坏死因子-α显著相关，R = 0.43。我们大鼠模型中的细胞因子反应模式在不同分析方法之间也存在差异，但总体而言，ELISA和CBA产生了相似的结果。为了进行方法间比较，我们使用ELISA和CBA检测了ELISA试剂盒提供的细胞因子标准品，以确定R值，发现所有测试的细胞因子的R值均大于0.90。结果发现，ELISA在标准曲线的低范围内更敏感，而微珠分析能够检测更高的蛋白质浓度，这将允许直接测量浓缩样本。ELISA和基于流式细胞术微珠的分析方法的绝对蛋白值之间缺乏一致性；在大多数情况下，后一种方法给出的蛋白质浓度往往高于ELISA。总之，在本研究中测试的分析方法之间，绝对蛋白值的直接比较不一致，但ELISA和CBA之间的细胞因子反应模式通常一致。对于小鼠CBA的情况，如果报告了低浓度分析物的样本并外推至低于灵敏度或零，则建议进行配套测量。
