The Monocyte Activation Test (MAT) is an in vitro assay for pyrogen testing in pharmaceutical and medical device industries. The principle of the Monocyte Activation Test is based on the detection of inflammatory cytokines released by human monocytes in response to exogenous bacterial endotoxin (LPS) and non-endotoxin pyrogens (NEP). Since its first adoption by the European Pharmacopeia in 2010 (Ph. Eur. chapter 2.6.30) the Monocyte Activation Test is recognized as a compendial method for pyrogen testing in several world regions including Europe. Here, the Monocyte Activation Test is recommended as a replacement test for the Rabbit Pyrogen Test (RPT) (1). The Monocyte Activation Test is compliant to the EU Directive 2010/63/EU on the protection of experimental animals and the global framework of the 3Rs (replacement, refinement, reduction of experimental animal tests). (2) The PyroCell® Monocyte Activation Test is performed in two steps: during an overnight cell culture step, the peripheral blood mononuclear cells (PBMC) preparation is stimulated for release of inflammatory cytokines with a product sample to be tested for the presence of pyrogens. On the next day, the cell culture supernatant is analyzed for released IL-6 cytokines using the PeliKine human IL-6 ELISA assay. The color reaction is measured in an absorbance reader, at a wavelength of 450 nm and a reference wavelength of 540-590 nm. The pyrogen 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 use of the PyroCell® MAT HS Kit : 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 190035 LAL Reagent GradeTM Multi-well Plates 25-340 Endotoxin-free accessories, i.e. sample container, pipette tips, serological pipettes See Catalog Step 2: IL-6 ELISA Assay Product Catalog # ELx808 Reader (with 450 and 540-590 nm filter) 25-315S The PyroCell® Monocyte Activation Test – Human Serum System (PyroCell® MAT HS System) is an all-inclusive kit containing validated reagents for the Monocyte Activation Test: the PyroCell Monocyte Activation Test – Human Serum Kit (PyroCell® MAT HS Kit) that contains the cryopreserved, human PBMC preparation pooled from 4 human blood donations (pMAT Cells) along with an optimized Human Serum Supplement, and the Pelikine compact human IL-6 ELISA kit that contains all reagents and buffers to conduct the IL-6 ELISA. pMAT Cells are specifically qualified for pyrogen testing with the Monocyte Activation test, e.g. are certified for their ability to detect endotoxins and non-endotoxin pyrogens. Thereby, donor selection, cell preparation and quality testing strictly follows compendial requirements. The Human Serum component is formulated with special attention to a low endotoxin content in order to ensure error-free cell stimulation conditions. The use of Human Serum in Monocyte Activation Tests has been demonstrated as beneficial especially for plasma derived products since less dilution is needed to overcome product mediated interferences. The Pelikine compact human IL-6 ELISA kit is composed of an ELISA detection kit and buffer concentrates ensuring the highly sensitive detection of the pro-inflammatory cytokine interleukin 6 (IL-6). The PyroCell® MAT HS System is the optimal tool for the sensitive detection of the IL-6 cytokine released by human monocytes during exposure to endotoxins or non-endotoxin pyrogens. All components together support the complete testing of 3x 96 well plates (288 tests) or up to 9 product samples according to the compendial requirements for Monocyte Activation Tests. The historical LOD of the PyroCell® MAT HS System is 0.08 EU/mL. (1) European Pharmacopeia (2) Global framework 3R
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