Monocyte Activation Test Meets Digital PCR
Dec 16, 2025 | Informative Articles
The MAT is based on the activation of monocytes by pyrogenic substances present in the sample. Upon exposure to potential pyrogens, monocytes undergo a signalling pathway resulting in secretion of pro-inflammatory cytokines, such as IL-1β, TNF-α and IL-6.
Implementation of MAT remains complex, often requiring experienced personnel, long-term cell stimulation, time-consuming steps and fresh donor blood or PBMCs. To address these challenges, Minerva Biolabs has developed the NAT-MAT® system—a next-generation pyrogen detection platform designed to offer a standardized, sensitive, and user-friendly alternative to conventional ELISA-based MAT setups.
The NAT-MAT® combines the established MAT with modern nucleic acid amplification technique (NAT), in fact digital PCR (dPCR). This system measures the presence of pyrogens by monitoring cytokine release from a standardized monocytic cell line. In contrast to traditional MAT assays that detect cytokine proteins using ELISA, the NAT-MAT® measures cytokine induction at the mRNA level, capturing earlier immune activation events and thus providing an earlier and more sensitive indication of pyrogenic activity.
The NAT-MAT® system relies on the use of NAT-MAT® Cells, which are stimulated with a pyrogen standard and the sample to be tested followed by nucleic acid extraction and subsequent dPCR analysis of the gene expression of IL-1β and TNF-α.
Due to its high degree of automation, the workflow reduces hands-on time to a minimum, significantly decreasing the need for manual operator involvement.
Does the NAT-MAT Detect the Same Phenomenon as ELISA-based MATs?
While the NAT-MAT® quantifies cytokine responses at the transcriptional level via digital PCR (dPCR), the assay targets the same innate immune signalling cascade as conventional ELISA-based MATs that measure protein expression. Upon activation of human monocytes by pyrogenic stimuli, pattern-recognition receptors—predominantly Toll-like receptors (TLRs)—trigger intracellular signalling pathways such as NF-kB and MAPK [1]. These cascades lead to the rapid transcriptional upregulation of key pro-inflammatory cytokine genes, including TNF-α, IL-1β and IL-6 followed by translation and secretion of the corresponding proteins.
Numerous studies in stimulated immune cells show that cytokine transcripts such as TNF-α and IL-1β are rapidly up-regulated (often within 1–2 h) and that protein secretion typically follows within a few hours. For example, single-cell analyses in THP-1 monocytes observed TNF-α mRNA peaking ~1 h and IL-1β ~2 h post-LPS [2]. Meanwhile, large‐scale transcriptomics vs. proteomics studies report moderate to strong mRNA/protein correlations for secreted cytokines under matched conditions [3] [4].
The resulting dose–response curves for TNF-α and IL-1β obtained by digital PCR correspond with those obtained by ELISA (see Figures 1 and 2). These parallel standard curves confirm that quantification at the mRNA level reflects the kinetics of cytokine protein expression and confirm that detection at the transcriptional level is biologically relevant. In NAT-MAT®, the detection of cytokine mRNA thus represents an earlier but equivalent indicator of monocyte activation.
Dose-response curves of IL-1β (top) and TNF-α (bottom) in NAT-MAT® Cells. Comparison of protein expression obtained by ELISA (left) and gene expression obtained by digital PCR (right).
By combining the biological relevance of the MAT with the precision and sensitivity of digital PCR, the NAT-MAT® system represents a significant step forward in pyrogen detection. While the underlying immunological mechanism remains the same as in conventional ELISA-based MATs, the NAT-MAT® shifts detection to the transcriptional level, capturing cytokine responses earlier in the activation cascade. This approach delivers sensitivity and robustness without compromising biological equivalence, as demonstrated by the parallel dose–response curves obtained with both ELISA and dPCR.
[1] https://www.cell.com/trends/molecular-medicine/fulltext/S1471-4914(07)00184-0 [2] https://www.nature.com/articles/s41598-021-92846-0.pdf [3] https://link.springer.com/article/10.1007/s11033-014-3585-8 [4] https://www.sciencedirect.com/science/article/pii/S0014579309008126