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Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α
Published on: June 14, 2018
A user-friendly, highly sensitive assay to detect the IFN-gamma secretion by T cells
Paul L A M Corstjens1, Michel Zuiderwijk, Hans J Tanke
1Leiden University Medical Center, Department of Molecular Cell Biology, Leiden, The Netherlands. Corstjens@LUMC.NL
Insights
A new lateral flow assay (ULIGA) offers a user-friendly alternative to ELISA for detecting interferon-gamma (IFN-gamma) from peripheral blood mononuclear cells (PBMC). This sensitive assay is suitable for single-sample testing with reduced equipment and labor demands.
Area of Science:
- Immunology
- Biotechnology
- Assay Development
Background:
- Interferon-gamma (IFN-gamma) is a critical cytokine in immune responses.
- ELISA-based assays are standard for IFN-gamma detection but can be labor-intensive.
- A need exists for simpler, user-friendly IFN-gamma detection methods.
Purpose of the Study:
- To develop a user-friendly lateral flow (LF) assay as an alternative to ELISA for detecting IFN-gamma.
- To utilize up-converting phosphor (UCP) reporter particles in the LF assay.
- To enable IFN-gamma detection from in vitro cultured peripheral blood mononuclear cells (PBMC).
Main Methods:
- Adapted molecular components of an IFN-gamma ELISA into a UCP-based LF immuno-sandwich assay (ULIGA).
- Determined the analytical sensitivity and dynamic range of the ULIGA.
- Validated the assay qualitatively using supernatants from PBMC cultures stimulated with M. leprae antigens, mitogen, or medium.
Main Results:
- ULIGA demonstrated analytical sensitivity better than 2 pg/mL.
- The assay exhibited a dynamic range spanning four orders of magnitude.
- ULIGA results showed good correlation with conventional IFN-gamma ELISA.
Conclusions:
- ULIGA can detect IFN-gamma levels well below the ELISA cutoff (100 pg/mL).
- The assay is less demanding in terms of equipment and labor compared to ELISA.
- ULIGA is well-suited for testing single samples efficiently.
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.
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