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重组C因子检测中批次间重复性及检测介质选择影响的评估。

Evaluation of lot-to-lot repeatability and effect of assay media choice in the recombinant Factor C assay.

作者信息

McKenzie Jennifer Helen, Alwis K Udeni, Sordillo Joanne E, Kalluri Kesava Srinivas, Milton Donald Kirby

机构信息

Biomedical Engineering and Biotechnology Program, University of Massachusetts, Lowell, MA, USA.

出版信息

J Environ Monit. 2011 Jun;13(6):1739-45. doi: 10.1039/c1em10035a. Epub 2011 May 9.

Abstract

Measurement of environmental endotoxin exposures is complicated by variability encountered using current biological assay methods arising in part from lot-to-lot variability of the Limulus-amebocyte lysate (LAL) reagents. Therefore, we investigated the lot-to-lot repeatability of commercially available recombinant Factor C (rFC) kits as an alternative to LAL. Specifically, we compared endotoxin estimates obtained from rFC assay of twenty indoor dust samples, using four different extraction and assay media, to endotoxin estimates previously obtained by Limulus amebocyte lysate (LAL) assay and amounts of 3-hydroxy fatty acids (3-OHFA) in lipopolysaccharide (LPS) using gas-chromatography mass spectroscopy (GC-MS). We found that lot-to-lot variability of the rFC assay kits does not significantly alter endotoxin estimates in house dust samples when performed using three of the four assay media tested and that choice of assay media significantly altered endotoxin estimates obtained by rFC assay of house dust samples. Our findings demonstrate lot-to-lot reproducibility of rFC assay of environmental samples and suggest that use of rFC assay performed with Tris buffer or water as the extraction and assay medium for measurement of endotoxin in dust samples may be a suitable choice for developing a standardized methodology.

摘要

环境内毒素暴露的测量因使用当前生物测定方法时遇到的变异性而变得复杂,部分原因是鲎试剂(LAL)的批次间变异性。因此,我们研究了市售重组C因子(rFC)试剂盒的批次间重复性,以替代LAL。具体而言,我们使用四种不同的提取和测定介质,比较了从20个室内灰尘样本的rFC测定中获得的内毒素估计值,与先前通过鲎试剂(LAL)测定获得的内毒素估计值以及使用气相色谱-质谱联用(GC-MS)测定的脂多糖(LPS)中3-羟基脂肪酸(3-OHFA)的含量。我们发现,当使用测试的四种测定介质中的三种进行rFC测定试剂盒的批次间变异性分析时,其不会显著改变室内灰尘样本中的内毒素估计值,并且测定介质的选择会显著改变通过rFC测定室内灰尘样本获得的内毒素估计值。我们的研究结果证明了rFC对环境样本测定的批次间可重复性,并表明使用Tris缓冲液或水作为提取和测定介质进行rFC测定以测量灰尘样本中的内毒素,可能是开发标准化方法的合适选择。

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