一种用于检测T细胞分泌γ干扰素的用户友好型高灵敏度检测方法。
A user-friendly, highly sensitive assay to detect the IFN-gamma secretion by T cells.
作者信息
Corstjens Paul L A M, Zuiderwijk Michel, Tanke Hans J, van der Ploeg-van Schip Jolien J, Ottenhoff Tom H M, Geluk Annemiek
机构信息
Leiden University Medical Center, Department of Molecular Cell Biology, Leiden, The Netherlands.
出版信息
Clin Biochem. 2008 Apr;41(6):440-4. doi: 10.1016/j.clinbiochem.2007.12.015. Epub 2008 Jan 3.
OBJECTIVES
Development of a user-friendly test alternative to ELISA-based assays to detect IFN-gamma by in vitro cultured peripheral blood mononuclear cells (PBMC) stimulated with pathogen-derived antigens.
DESIGN AND METHODS
The molecular components of an operational IFN-gamma ELISA-based test were applied in a lateral flow (LF) immuno-sandwich assay using up-converting phosphor (UCP) reporter particles. The analytical sensitivity of the UCP-LF IFN-gamma assay (ULIGA) was determined and the assay was qualitatively validated with a selection of 60 supernatants derived from PBMC cultures stimulated with M. leprae derived antigens, mitogen or medium alone.
RESULTS
ULIGA indicated an analytical sensitivity better than 2 pg/mL, and demonstrated four orders of magnitude dynamic range. The assay correlated well with the IFN-gamma ELISA.
CONCLUSIONS
ULIGA allows detection well below the cutoff value (100 pg/mL) used to define positive responses in the IFN-gamma ELISA. The test procedure is less demanding in respect to equipment and labor, and is suited for testing single samples.
目的
开发一种用户友好的检测方法，以替代基于酶联免疫吸附测定（ELISA）的方法，用于检测经病原体衍生抗原刺激的体外培养外周血单个核细胞（PBMC）产生的γ干扰素（IFN-γ）。
设计与方法
将基于ELISA的可操作IFN-γ检测的分子成分应用于使用上转换磷光体（UCP）报告颗粒的侧向流动（LF）免疫夹心测定中。测定了UCP-LF IFN-γ测定法（ULIGA）的分析灵敏度，并用60种分别来源于经麻风分枝杆菌衍生抗原、丝裂原或仅培养基刺激的PBMC培养物的上清液进行了定性验证。
结果
ULIGA显示出优于2 pg/mL的分析灵敏度，并展示了四个数量级的动态范围。该测定法与IFN-γ ELISA相关性良好。
结论
ULIGA能够检测到远低于用于定义IFN-γ ELISA阳性反应的临界值（100 pg/mL）。该检测方法对设备和人力的要求较低，适合单个样本的检测。
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