Introducing NAT-MAT^®: Fast and Precise Pyrogen Detection Using Digital PCR
With increasing regulatory focus on animal-free methods, the Monocyte Activation Test (MAT) has gained traction as a replacement of the Rabbit Pyrogen Test (RPT). Yet its implementation remains complex, often requiring experienced personnel, time-consuming steps and fresh donor blood or pooled 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.

What is NAT-MAT^®?
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.

The NAT-MAT^® system is an in vitro assay that measures the presence of pyrogens by monitoring cytokine release from a standardized monocytic cell line. In contrast to conventional MAT kits, that measure the protein secretion of cytokines using ELISA, the NAT-MAT^® measures the gene expression of IL-1β and TNF-α using nucleic acid amplification technique (NAT), in fact digital PCR technology.

What makes the NAT-MAT^®…

…more accurate?
A special feature of the NAT-MAT^® is the normalization of the pyrogen concentration to the cell number, that might vary from well to well. This is possible by parallelly measuring the gene expression of the cytokines and the gene expression of a housekeeping gene. Normalizing the pyrogenic load to the cell number leads to highly accurate results and dependable data.

… faster?
ELISA-based MATs measure the protein expression of the cytokine and require overnight stimulation of the cells. In contrast, the NAT-MAT^® is based on the measurement of the cytokine´s gene expression. For this reason, the cells only need to be stimulated for 4 hours.

… more reliable?
Conventional MAT kits often use PBMCs as a cell source, which can vary widely from lot to lot, leading to inconsistent results. In addition, cell supply with PBMCs can be a challenge.

The NAT-MAT^® uses a stable cell line of HL-60 derived macrophages, which ensures reproducible outcomes and a reliable cell supply.

… save your time?
The NAT-MAT^® can be highly automated. The extraction process and pipetting can be carried out automatically by robots, which saves time and reduces manual steps to a minimum.  

… more robust?
Existing MATs are based on the measurement of only one cytokine. The NAT-MAT^® measures the gene expression of two cytokines – IL-1β and TNF-α. This enables sensitive and robust quantification across varying pyrogen levels.

How It Works?

1. Seeding: Seed ready-to-use, cryopreserved HL-60 derived macrophages over night
2. Stimulation: Stimulate cells for 4 hours
3. Extraction: Extract nucleic acids with magnetic-bead-based extraction kit
4. Detection: Analyse the gene expression of IL-1β, TNF-α and a housekeeping gene via dPCR
5. Data Analysis: Calculate pyrogen load in the test sample with an Excel-based analysis template or an analysis software

Why it matters – the top advantages

• Using a stable cell line ensures reproducible outcomes and a reliable cell supply
• Normalizing the pyrogen concentration to the cell number delivers highly precise results
• Save time with rapid stimulation of only 4 hours
• The highly automatable workflow saves time and reduces hand-on time to a minimum
• Dual-cytokine detection increases dynamic range and enhances assay robustness
• Data analysis with excel analysis template or easy-to-use software

With the ongoing revision of global pharmacopeias, there are more and more efforts to abolish animal-based methods like the RPT.  The NAT-MAT^® aligns with this shift, offering a validated alternative designed to support modern, animal-free pyrogen testing.

Since the RPT will be discontinued in Europe, regulatory authorities currently encourage manufacturers to implement MAT in their QC system. Get a head start on RPT transition and rely on NAT-MAT^®!