A novel ELISpot method for adherent cells

Journal of Immunological Methods
Chantal K TurnerRichard P Phipps

Abstract

The purpose of this study was to develop an enzyme-linked immunospot assay (ELISpot assay) that can be used with human adherent cells. While standard enzyme-linked immunosorbent assays (ELISAs) are available and widely used and ELISpot assays are used for nonadherent lymphocytes, no ELISpot assay has been developed for adherent cells. We used primary human fibroblasts from four different tissues (myometrium, lung, gingiva, and orbit), either unstimulated or interleukin (IL)-1beta-activated, to evaluate an ELISpot assay. Antibody pairs for IL-6 and IL-8 were used and results were compared to a standard ELISA. We found that we could reliably detect IL-6 and IL-8 spots with as few as 10 fibroblasts. Optimal cell numbers were 50 cells per well incubated for 8 h, although spots appeared as early as 2 h after incubation. Spots were absent when cells, primary, or secondary anti-cytokine antibodies were omitted from the protocol. Spot number and size can be ascertained using current automated ELISpot reader technology. The frequency of IL-6 and IL-8-producing human fibroblasts could also be determined. For example, 60% of the lung fibroblasts express IL-6, but IL-8 can be detected from only 40% of the cells. Approximately 80% of the hu...Continue Reading

References

Dec 16, 1983·Journal of Immunological Methods·C C CzerkinskyA Tarkowski
Oct 1, 1995·Clinical Immunology and Immunopathology·K M FriesR P Phipps
Dec 1, 1994·Cell and Tissue Research·R C Henrikson, T J Smith
Jun 6, 2000·Journal of Immunotherapy·A SchmittelC Scheibenbogen
Jun 16, 2000·Journal of Immunological Methods·F H RininslandM Tary-Lehmann
Jul 11, 2002·American Journal of Physiology. Cell Physiology·Laura Koumas, Richard P Phipps
Oct 25, 2002·Diabetes/metabolism Research and Reviews·Guido MeierhoffNanette C Schloot

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