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PyroDetect System - The Human(e) Alternative in Pyrogen Testing: Monocyte-Activation Test using cryo-blood and Interleukin-1β response

Merck:/topic/images/BioM/pyrodetect_plates_key_visual_177.jpgThe PyroDetect System is a revolutionary test for the detection of pyrogens based on the Monocyte-Activation Test (MAT).  The PyroDetect Sytem uses an innate immune defense reaction.  Monocytes activated by pyrogens produce cytokines that are detected in an immunological assay (ELISA) involving specific antibodies and an enzymatic color change.

  • Broad Pyrogen Profile
  • Easy Assay Procedure
  • Robust Application

The Monocyte-Activation Test (MAT) was introduced in the European Pharmacopoeia  in 2010 as an alternative to the rabbit test (EP Chapter 2.6.30). Using whole human blood (cryo-blood or fresh blood), the PyroDetect System mimics the innate immune response to a fever reaction caused by pyrogens.  The MAT test simulates the human fever reaction better than the Rabbit pyrogen test or the LAL test.
The PyroDetect System offers a high level of safety using both a positive and a negative control. The robust application of the test was shown in an international validation coordinated by the European Center for the Validation of Alternative Methods (ECVAM) in 2005. Several published studies show the robustness and reliability of the test performance of the MAT.

The PyroDetect System mimics the innate immune response in humans


Assay procedure of the PyroDetect System

Easy Assay Procedure
The PyroDetect System consists of three main steps, the cryoblood (or fresh blood) incubation, the IL-1β ELISA, and the analysis.

Cryo-blood Incubation
The PyroDetect System uses cryo-blood (pooled and frozen
human whole blood) for the blood incubation. The test samples are mixed with the blood in the cell culture plate and are kept in an incubator over night at 37 °C. If pyrogens are present in the sample, the monocytes of the blood will produce IL-1β during the incubation.

For the detection of the IL-1β the cryo-blood incubation
mixture is transferred into an ELISA microplate coated with an antibody specific for IL-1β. Interleukin molecules present are bound by the immobilized antibody. Then an enzyme-linked polyclonal antibody and a substrate is added. The following color reaction allows the detection of the bound IL-1β in an ELISA reader.

The pyrogen concentration in the sample is then determined
from the IL-1β concentration via an endotoxin standard curve.


For the last decade, the rabbit test and the Limulus Amebocyte Lysate (LAL) test were used for the detection of pyrogens. Both methods are limited in the products which can be analysed and both have a high level of animal consumption. In the case of the LAL test, only endotoxins can be detected causing a safety risk. To overcome these limitations, the Monocyte- Activation Test (MAT) was introduced in the European Pharmacopoeia in 2010.



Comparison of Pyrogen Test Methods
  Test principle Fever reaction in mammal Defence mechanism  Fever reaction in human 
    Contamination Gram-negative (LPS) +  + + 
Gram-positive (LTA) + - +
Yeasts & Molds + - +
Virus +/-1 - +
    Application Pharmaceuticals + + +
Biologicals (e.g. gene therapy, recombinant therapeutic proteins) + +/-2 +
Medical devices +3 +/-3 +
Cell therapeutics (e.g. monoclonale antibody) - +/-  +
 1 Variable pyrogen results, 2 Rabbit test often required, 3 indirect test with solution in pyrogen-free water   

Table 1: Comparison of the Rabbit Test, the LAL Test and the PyroDetect Test

Now also available: NEW PyroDetect Data Analysis Tool

Ordering Information

The PyroDetect System consists of the PyroDetect Kit (Art. No. 144 154) and the PyroDetect Cryoblood (Art. No. 144 155). Except for the endotoxin standard it contains all biological and biochemical reagents required for the MAT. To perform the test an endotoxin standard, the PyroDetect Endotoxin Standard (Art. No. 144 161) or an equivalent international reference standard (RSE) can be used. The PyroDetect Interleukin Standard (Art. No. 144 158) is optional and can be used for additional control reactions in the Interleukin-1β ELISA.


PyroDetect Kit 1.44154.0001 Up to 4 tests
PyroDetect Cryoblood 
1.44155.0001 2 x 2ml
PyroDetect Interleukin Standard
1.44158.0001 Up to 10 reactions
PyroDetect Endotoxin Standard 1.44161.0001 1 vial
PyroDetect Data Analysis Tool 1.44299.0001 1 CD


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