The fever reaction results from monocyte activation through microbial products. The Monocyte Activation Test (MAT) detects this reaction in vitro, as outlined here. Briefly, when performing MAT, PBMC (that have been cryopreserved instantly after their isolation form the human body) are exposed in tissue culture to the test substance. If the test substance contains microbial contaminants, these will elicit IL-6 production in the monocytes, which can be detected by an IL-6 ELISA in the culture supernatant.

Representing a sensitive in-vitro bioassay, successful implementation of MAT critically depends on the availability of fully functional monocytes as indicator cells, optimized and pyrogen-free tissue culture conditions, and a sensitive cytokine detection in the culture supernatant. Our experts are available to perform and interpret test results, including product-specific validation and interference matrices, all while documenting and reporting the test procedure to meet regulatory compliances. CTL-MAT is the only company that can help you with any and all these requirements for performing the MAT at your own facility – or we can perform the test for you in our own certified laboratories.

People throughout the world should be able to count on parenteral drugs, cosmetics and biopharmaceuticals to be safe. The same holds true for medical devices. Microbial contamination, however, can happen even under the most stringent manufacturing standards.

Such contaminations can lead to conditions ranging from mild fever to life-threatening septic shock in recipients. This is why regulatory agencies throughout the world mandate "pyrogenicity testing" of every production batch of parenteral drugs and medical devices.

A product's pyrogenicity due to contamination, that is, its property to induce a fever reaction has been tested traditionally by injecting it into rabbits, in the Rabbit Pyrogen Test, RPT. While the RPT is ideally suited to detect microbial contaminants of all kinds (constituents of Gram-positive and Gram-negative Bacteria, viruses, fungi and mold), it involves animal testing (see below).

As the mechanism of this fever reaction is in the meantime well understood, it has become possible to model it in cell culture experiments in vitro via the Monocyte Activation Test (MAT) the principle of this test is described here  As MAT has been proven to be a superior detection system for microbial contaminations compared to the RPT, however without involving animal sacrifice. The European Pharmacopeia has mandated to avoid the RPT whenever possible in favor of in vitro laboratory testing, and in Ph. Eur. 2.6.30 has come up with strict guidelines for the implementation, product-specific validation and reporting for MAT.

MAT has additional advantages over RPT: It's applicable to a greater variety of products than RPT, and furthermore it is more precise, reliable, and cost-effective compared to RPT.

CTL-MAT's Test kit and customer support structure has been established to help realize the goal of maximal drug safety avoiding unnecessary animal cruelty.

White blood cells of the North American horse shoe crab, limulus, contain a protein called Factor C, that agglutinates in the presence of lipopolysaccharides (LPS). Relying on this reaction, the Limulus Amebocyte Lysate test (LAL) has emerged as an in vitro alternative for the RPT to detect contaminations with LPS. The test principle of the LAL test is described here Unfortunately, at least one third of the crabs do not survive the bleeding. To avoid unnecessary animal cruelty, the trend is to obtain Factor C not by bleeding of these animals, but by production as a recombinant protein. Irrespective of Factor C's origin, the test principle is the same resulting in a highly sensitive and specific tests for the detection of LPS (and LPS only -other microbial contaminants equally hazardous go undetected (see below). LPS is contained in Gram-negative bacteria, but not in Gram-positive bacteria, in fungi and viruses. Therefore, LAL and Factor C tests fail to detect all these contaminations, except for LPS.

Pyrogens detected by the Rabbit Pyrogen Test (RPT), by the Monocyte Activation Test (MAT)

MAT detects all the above microbial contaminants. Monocytes rely on a family of pattern recognition receptors, called toll-like receptors (TLR) to identify such targets and to eliminate them by phagocytosis. LPS engages TLR-4 on monocytes, triggering them to produce IL-6 (thus, like LAL, MAT also detects LPS). Lipoteichoic acid (LTA), a constituent of Gram-positive bacteria, triggers TLR-2 on monocytes, as does peptidoglycan (PGN), a constituent of most bacteria, or Zymosan, a constituent of yeast). By expressing 9 different types of TLRs as known today, monocytes are well equipped to recognize most microbial agents. For a summary of TLRs and their respective microbial ligands (see here)

Unlike the LAL test (and recombinant Factor C assay), MAT is uniquely suited to detect microbial contaminations of all kinds beyond LPS. Representative data showing detection of key microbial contaminants beyond LPS by MAT are shown here.

Like the MAT, the LAL and the rFC are compendial methods, i.e., they need to undergo product-specific validation. As the MAT is accepted as an animal- free quantifiable method for pyrogen testing, the Eu. Ph. requires MAT, instead of the RPT to be used for the validation of LAL and rFC.