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Using PBMCs in a Multiplex FluoroSpot Assay for Detection of Innate Immune Response-Modulating Impurities (IIRMIs)
Bartek Makower1, Niklas Ahlborg2,3
1Mabtech AB, Nacka Strand, Sweden. bartek.makower@mabtech.com.
Methods in Molecular Biology (Clifton, N.J.)
|March 19, 2024
Summary
The monocyte activation test (MAT) offers a human-based alternative to the rabbit pyrogen test (RPT) for detecting impurities in drugs. A FluoroSpot assay quantifies activated monocytes and pyrogenic cytokines, improving safety assessments.
Area of Science:
- Immunology
- Pharmacology
- Biotechnology
Background:
- The rabbit pyrogen test (RPT) is traditionally used to detect pyrogens in injectable drugs.
- Innate Immune Response-Modulating Impurities (IIRMIs) can trigger pyrogenic reactions.
- There is a need for alternative, human-based testing methods.
Purpose of the Study:
- To evaluate the monocyte activation test (MAT) as a replacement for the RPT.
- To utilize a triple-color FluoroSpot assay for sensitive detection of IIRMI-activated monocytes and cytokines.
Main Methods:
- Human peripheral blood mononuclear cells (PBMCs) were used.
- An ELISA-based monocyte activation test (MAT) was employed.
- A triple-color IL-1β/IL-6/TNF-α FluoroSpot assay was developed and utilized.
Main Results:
- The FluoroSpot assay effectively quantified frequencies of IIRMI-activated monocytes.
- The assay determined the relative amounts of pyrogenic cytokines (IL-1β, IL-6, TNF-α) produced by activated cells.
- This method provides a sensitive tool for impurity detection.
Conclusions:
- The MAT, using a FluoroSpot assay, is a viable and sensitive alternative to the RPT for detecting IIRMIs.
- This human-based approach enhances the safety assessment of injectable drugs.
