Optimising the quantification of cytokines present at low concentrations in small human mucosal tissue samples using Luminex assays.
Optimising the quantification of cytokines present at low concentrations in small human mucosal tissue samples using Luminex assays.
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DOI：
10.1016/j.jim.2013.04.009
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发表时间：
2013-08-30
影响因子：
2.2
通讯作者：
Robinson, Karen
中科院分区：
文献类型：
--
作者：
Staples, Emily;Ingram, Richard James Michael;Atherton, John Christopher;Robinson, Karen
从小的粘膜组织活检中敏感地测量多种细胞因子谱，例如通过内窥镜获得的人类胃活检组织，在技术上是具有挑战性的。Luminex分析等多重方法提供了一个有吸引力的解决方案，但标准方案不适用于组织样本。我们评估了三种商业Luminex试剂盒(VersaMAP、Bio-Plex和MILLIPLEX)在测量人胃活检组织中白细胞介素17A(IL-17)和干扰素-γ(干扰素γ)浓度的有效性，并针对这一应用优化了粘膜样品的制备。首先，我们评估了每个试剂盒的技术性能、灵敏度极限和线性动态范围。接下来，我们用重组IL-17和干扰素γ在一定范围内(1.5至1000npg/mL)添加人胃活检组织，并评估试剂盒在添加细胞因子回收率和检测内精密度方面的准确性。我们还评估了不同的组织处理方法和提取缓冲液对结果的影响。最后，我们评估了未添加添加剂的样品中内源性细胞因子的回收率。在敏感性方面，所有试剂盒都在制造商推荐的标准曲线范围内表现良好，但MILLIPLEX试剂盒在低细胞因子浓度下提供了最一致的敏感性。在添加实验中，MILLIPLEX试剂盒在最广泛的浓度范围内表现最稳定。对于组织处理，与自动化方法相比，人工破坏提供了显著改善的细胞因子恢复。我们选择的试剂盒和优化的方案通过使用Luminex和实时聚合酶链式反应测量炎症和非炎症胃粘膜中的相对细胞因子水平来进一步验证。总之，通过适当的优化，Luminex试剂盒(特别是针对IL-17和干扰素γ的MILLIPLEX试剂盒)可以用于灵敏地检测粘膜活检中的细胞因子。我们的结果应该会帮助其他研究人员对小组织样本中的多种低浓度细胞因子进行量化。挑战：使用小粘膜组织样本溶液评估体内细胞因子谱：低容量多重分析和优化的样品制备我们比较了三种Luminex试剂盒对低浓度IL-17和干扰素γ的定量。MILLIPLEX在敏感性和准确的细胞因子回收方面最为一致。人工组织处理和PBS提取缓冲液效果较好。
Sensitive measurement of multiple cytokine profiles from small mucosal tissue biopsies, for example human gastric biopsies obtained through an endoscope, is technically challenging. Multiplex methods such as Luminex assays offer an attractive solution but standard protocols are not available for tissue samples. We assessed the utility of three commercial Luminex kits (VersaMAP, Bio-Plex and MILLIPLEX) to measure interleukin-17A (IL-17) and interferon-gamma (IFNγ) concentrations in human gastric biopsies and we optimised preparation of mucosal samples for this application. First, we assessed the technical performance, limits of sensitivity and linear dynamic ranges for each kit. Next we spiked human gastric biopsies with recombinant IL-17 and IFNγ at a range of concentrations (1.5 to 1000 pg/mL) and assessed kit accuracy for spiked cytokine recovery and intra-assay precision. We also evaluated the impact of different tissue processing methods and extraction buffers on our results. Finally we assessed recovery of endogenous cytokines in unspiked samples. In terms of sensitivity, all of the kits performed well within the manufacturers' recommended standard curve ranges but the MILLIPLEX kit provided most consistent sensitivity for low cytokine concentrations. In the spiking experiments, the MILLIPLEX kit performed most consistently over the widest range of concentrations. For tissue processing, manual disruption provided significantly improved cytokine recovery over automated methods. Our selected kit and optimised protocol were further validated by measurement of relative cytokine levels in inflamed and uninflamed gastric mucosa using Luminex and real-time polymerase chain reaction. In summary, with proper optimisation Luminex kits (and for IL-17 and IFNγ the MILLIPLEX kit in particular) can be used for the sensitive detection of cytokines in mucosal biopsies. Our results should help other researchers seeking to quantify multiple low concentration cytokines in small tissue samples. Challenge: To evaluate in vivo cytokine profiles using small mucosal tissue samples Solution: Low volume multiplex assays and optimised sample preparation We compared low concentration IL-17 and IFNγ quantification by three Luminex kits. MILLIPLEX was most consistent for sensitivity and accurate spiked cytokine recovery. Manual tissue processing and PBS-based extraction buffers were superior.
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影响因子：
--
作者：
Pickering, Jerry W.;Larson, Matthew T.;Hill, Harry R.
通讯作者：
Hill, Harry R.
DOI：
10.1046/j.1440-1746.1999.01810.x
发表时间：
1999-01-01
影响因子：
4.1
作者：
Kusugami, K;Ando, T;Kaneko, H
通讯作者：
Kaneko, H
影响因子：
3.1
作者：
Lindholm, C;Quiding-Järbrink, M;Svennerholm, AM
通讯作者：
Svennerholm, AM
影响因子：
4.5
作者：
Luzza, F;Parrello, T;Pallone, F
通讯作者：
Pallone, F
影响因子：
3
作者：
Houser B
通讯作者：
Houser B