The Monocyte Activation Test (MAT) is an in vitro assay for pyrogen testing in the biopharmaceutical or medical device industry. The MAT test principle is based on the detection of inflammatory cytokines released by human monocytes in response to exogenous endotoxins (LPS) and non-endotoxin pyrogens (NEP). Since its first adoption into the European Pharmacopeia in 2010 (Chapter 2.6.30) the MAT is recognized as a compendial method for pyrogen testing in a number of world regions including Europe. Here, the MAT is recommended as a replacement test for the Rabbit Pyrogen Test (RPT) (1). The MAT is further compliant to the global framework of the 3Rs principle (replacement, refinement, reduction of experimental animal tests).(2)
The MAT assay is performed in two steps: during an overnight cell culture step, a monocytic cell preparation, e.g. peripheral blood mononuclear cells (PBMC) is stimulated for release of inflammatory cytokines with a product preparation that may contain a pyrogenic contaminant. On the next day, the cell culture supernatant is analyzed for the presence of released cytokines using an ELISA assay. The color reaction is measured in an absorbance reader, e.g. the ELx808 Reader, at a wavelength of 450 nm and a reference wavelength of 540-590 nm. The pyrogenic content of the sample is then analyzed against the reference standard endotoxin (RSE) and is expressed in Endotoxin Equivalent Units (EEU/mL).
The following reagents, accessories and instruments support the PyroCell™ MAT System, IL-6:
Step 1: Monocyte Activation
Product Catalog # Iscove's Modified Dulbecco's Medium (IMDM) with L-Glutamine and HEPES 12-722F USP Reference Standard Endotoxin E700 LAL Reagent Water W50-100 Pyrogen-free Dilution Tubes N207 Reagent Reservoirs 00190035 LAL Reagent Grade™ Multi-well Plates 25-340 Endotoxin-free accessories, i.e. sample container, pipette tips, serological pipettes See Catalog
Step 2: IL-6 ELISA
Product Catalog # ELx808 Reader (with 450 and 540-590 nm filter) 25-315S Endotoxin-free accessories, i.e. sample container, pipette tips, serological pipettes See Catalog
The PyroCell™ MAT System, IL-6 is an all-inclusive kit containing all necessary reagents for the MAT assay: the PyroCell™ MAT Kit that contains the cryopreserved PBMC preparation pooled from 4 healthy donors (pMAT Cells) along with the MAT Culture Medium Supplement, and the PeliKine compact human IL-6 ELISA kit that contains all reagents and buffer concentrates to conduct an IL-6 ELISA. The pMAT Cells are specifically qualified for endotoxins and non-endotoxin pyrogen testing with the MAT. Thereby, donor selection, cell preparation and quality testing strictly follow the requirements outlined in the Ph. Eur. Chapter 2.6.30. The MAT Culture Medium Supplement is formulated with special attention to a low endotoxin content in order to ensure error-free cell stimulation conditions. The PyroCell™ MAT Kit has been validated for use with the PeliKine compact human IL-6 ELISA kit for sensitive detection of the released pro-inflammatory cytokine interleukin 6 (IL-6). Therefore, the PyroCell™ MAT system is the optimal tool for the sensitive detection of the IL-6 cytokine released by human monocytes during exposure to endotoxins or pyrogens. All components together support the complete testing of 3x 96 well plates or up to 9 product samples according to the compendial requirements for the MAT assay. The historical LOD of the PyroCell™ MAT System, IL-6 is 0.02 EU/ml.
(1) European Pharmacopeia (2) Global framework 3R
The next chapter in endotoxin testing... Let's write it together.