The Monocyte Activation Test (MAT): A relevant alternative to the compendial sterility test
Introduction
Focus on the distinction between pathogen and non-pathogen microbes often overlooks the fact that residue from microbial necrosis can cause infection even in the case of microbes that are normally non-pathogenic. Furthermore, the widespread use of endotoxin detection has obscured the risks surrounding non-endotoxin pyrogens. The sterility test does not address the negative impact of dead bacteria and fungi. The monocyte activation test, already adopted by the European Pharmacopeia to replace the rabbit pyrogen test as a compendial method, extends the capacities of microbial detection to include non-endotoxin pyrogens.
Aim
To determine the range of pyrogen contaminants detected by the MAT
To evaluate the MAT sensitivity to heat-killed bacteria and fungi
Principle of the Monocyte Activation Test
Detection and quantification of cytokines by ELISA
Methods
Peripheral blood mononuclear cells (PBMC) pooled from 8 healthy adult donors adjusted to 106 cells/mL were incubated with serial dilutions of ligands for Toll-like receptors (TLR1-9) in 96-well microplates in 1:1 volume, for 20 hours at 37°C with 5% CO2. Pro-inflammatory interleukin-6 (IL-6), a pyrogenic biomarker, was evaluated by automated ELISA using the Simple Plex kits on Ella.
Heat killed Candida albicans and Listeria monocytogenes, at 109 cfu were resuspended in PBS then diluted with proprietary culture medium MAJIF1 from 107 to 102 cfu/mL. Pooled PBMC were incubated with serial dilutions of heat-killed C. albicans and L. monocytogenes, as described above.
Results
PBMC activation by TLR1-9 ligands resulted in significant IL-6 release, with the concentrations ranging from 3 ng/mL for TLR1 ligand pam3CSK4 to 156 ng/mL for TLR6 ligand FSL-1.
The USP reference standard endotoxin, known as a TLR4 ligand, was detected at concentrations as low as 0.002 EU/mL.
* Lysates of heat-killed C. albicans and L. monocytogenes at 10cfu/100µL also triggered a significant release of IL-6 with p-value <0.05. Dotted lines are levels of basal secretion of IL-6.
Conclusion
The pooled PBMC response to all TLR agonists suggests that pyrogens interacting with TLR other than TLR4 can be effectively detected with the monocyte activation test.
The detection of 10 cfu/100 µL of heat-killed fungus C. albicans or Gram-positive L. monocytogenes shows that the MAT is an effective alternative to the compendial sterility test, specifically useful for microbial slow growers and invasive pathogens.
About Pyrodex LLC
PyroDex LLC was developed by Djikolngar Maouyo, Ph.D., who has more than 25 years of experience in the areas of academic biomedical research and industrial biotechnology. He has focused exclusively on microbial detection for the past nine years and worked intensively in the development of various microbial detection kits while researching nascent technologies for microbial detection in the U.S. and Europe. Prior to founding PyroDex, Dr. Maouyo also held research faculty positions at the University of Maryland in Baltimore and the Johns Hopkins University School of Medicine. He now offers pyrogen and endotoxin testing services around the globe.