Development and validation of a multiplex microsphere-based assay for detection of domestic cat (Felis catus) cytokines.
Development and validation of a multiplex microsphere-based assay for detection of domestic cat (Felis catus) cytokines.
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开发和验证基于多重微球的检测方法，用于检测家猫（Felis catus）细胞因子。
DOI：
10.1128/cvi.00289-10
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发表时间：
2011
期刊：
影响因子：
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通讯作者：
Vandewoude,Sue
中科院分区：
文献类型：
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作者：
Wood,BrittaA;O'Halloran,KevinP;Vandewoude,Sue
细胞因子是介导先天免疫反应的重要信号分子，因此它们的存在具有诊断、预后和致病意义。基于微球的免疫分析可以快速准确地评估包括人类、狗和小鼠在内的几种物种的细胞因子水平；然而，评估家猫（Felis catus）细胞因子的技术一直局限于单分析物酶联免疫吸附试验（elisa）。基于微球的免疫测定提供了一种有吸引力的替代技术，可在单次测定中检测和定量多种分析物，仅需50 μl的样品。我们描述了一种基于微球的检测方法的开发和验证，该方法使用市售elisa试剂检测三种常用的家猫细胞因子（γ干扰素、白细胞介素-10和白细胞介素-12/白细胞介素- 23p40）。该方法对捕获和检测抗体浓度、链亲和素-藻红蛋白浓度和微球数量进行了优化。γ干扰素的检测下限和上限分别为31和1000 pg/ml，白介素-10的检测下限分别为63和2000 pg/ml，白介素-12/白介素- 23p40的检测下限分别为39和625 pg/ml。测定外周血单个核细胞上清液中的细胞因子浓度，微球法测定的结果与市售ELISA试剂盒测定的结果相关。该技术是一种方便、可重复的方法，可用于评估各种疾病引起的家猫细胞因子反应。
Cytokines are essential signaling molecules that mediate the innate immune response, and therefore their presence can be of diagnostic, prognostic, and pathogenic significance. Microsphere-based immunoassays allow rapid and accurate evaluation of cytokine levels in several species, including humans, dogs, and mice; however, technology to evaluate domestic cat (Felis catus) cytokines has been limited to single-analyte enzyme-linked immunosorbent assays (ELISAs). Microsphere-based immunoassays provide an attractive alternative technology for detecting and quantifying multiple analytes in a single assay using as little as 50 μl of sample. We describe the development and validation of a microsphere-based assay for three commonly analyzed domestic cat cytokines (gamma interferon, interleukin-10, and interleukin-12/interleukin-23 p40) using reagents from commercially available ELISAs. The assay was optimized for capture and detection antibody concentrations, streptavidin-phycoerythrin concentration, and number of microspheres. The validated lower and upper quantitation limits were 31 and 1,000 pg/ml for gamma interferon, 63 and 2,000 pg/ml for interleukin-10, and 39 and 625 pg/ml for interleukin-12/interleukin-23 p40. Cytokine concentrations in peripheral blood mononuclear cell supernatants were measured, and results obtained by the microsphere assay were correlated with values obtained with commercially available ELISA kits. This technology is a convenient and reproducible assay to evaluate domestic cat cytokine responses elicited by a variety of diseases.