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

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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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