A Simplified ELISpot Assay Protocol Used for Detection of Human Interleukin-4, Interleukin-13 and Interferon-γ Production in Response to the Contact Allergen Nickel

J. Minang, N. Ahlborg, M. Troye-Blomberg
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引用次数: 10

Abstract

Background: The ELISpot assay is a potential tool for in vitro diagnosis of contact allergy to nickel (Ni2+). A reduction of the assay time and work should further facilitate the development of ELISpot-based clinical and diagnostic applications. Objective: It was the aim of this study to evaluate a simplified ELISpot protocol utilizing plates precoated with capture monoclonal antibody (mAb) and one-step detection by an enzyme-labelled mAb. Methods: The frequency of Ni2+-induced IFN-γ-, IL-4- or IL-13-producing cells in human peripheral blood mononuclear cells from Ni2+-reactive and non-reactive subjects was determined. The simplified ELISpot was performed in parallel with a regular ELISpot assay utilizing overnight adsorption of capture mAb and detection in 2 steps. Results: Ni2+-induced IL-4 and IL-13 production was significantly greater in Ni2+-reactive subjects compared to the controls. The number of antigen-specific, cytokine-producing cells determined by the 2 different ELISpot protocols correlated well. Conclusion: The simplified ELISpot protocol provides a more rapid and easy alternative to the regular ELISpot, with a similar detection sensitivity for antigen-specific T-cell responses.
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一种用于检测人对接触性变应原镍产生的白细胞介素-4、白细胞介素-13和干扰素-γ的简化ELISpot检测方案
背景:ELISpot法是体外诊断镍(Ni2+)接触性过敏的潜在工具。减少分析时间和工作将进一步促进基于elispot的临床和诊断应用的发展。目的:本研究的目的是评估一种简化的ELISpot方案,该方案利用预先包被捕获的单克隆抗体(mAb)的板和酶标记的mAb一步检测。方法:测定Ni2+反应性和非反应性人外周血单核细胞中产生IFN-γ-、IL-4-和il -13细胞的频率。简化ELISpot与常规ELISpot平行进行,利用隔夜吸附捕获mAb并分2步检测。结果:Ni2+诱导的IL-4和IL-13的产生在Ni2+反应的受试者中显著高于对照组。通过两种不同的ELISpot方案测定的抗原特异性细胞因子产生细胞的数量相关性良好。结论:与常规ELISpot相比,简化的ELISpot方案提供了一种更快速、更简便的替代方案,对抗原特异性t细胞反应的检测灵敏度相似。
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Acknowledgement to Referees for Exogenous Dermatology 2004 Contents Vol. 3, 2004 Author Index Vol. 3, 2004 Is There Evidence that Geraniol Causes Allergic Contact Dermatitis? Subject Index Vol. 3, 2004
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