man whole blood, as a source of monocytes. The monocytes activated by pyrogens produce cytokines which
are detected in an enzyme-linked immunosorbent assay
The MAT employing fresh or cryo-preserved human
blood was described and validated as an in vitro pyrogen test [ 2, 3]. It has been included in the European
Pharmacopoeia (EP) since April 2010 [ 4].
For cryoblood incubation, sample or endotoxin are
mixed with cryoblood in a cell culture plate and kept
in an incubator at 37°C for 8-24 hours for interleukin-1ß
For the IL-1ß detection, the cryoblood incubation mixture is transferred into an ELISA plate coated with a mono-clonal antibody specific to IL-1ß. Interleukin molecules
present in the culture supernatant are bound by the immobilized antibody. After removal of unbound components
by several washing steps, an enzyme-linked polyclonal
antibody specific for IL-1ß is added. With the addition of
the substrate, a color reaction is started, which allows the
detection of the bound IL-1ß in an ELISA reader.
The pyrogen concentration in the sample is then determined from the IL-1ß concentration via an endotoxin
standard curve, and analyzed with the PyroDetect data
The broad pyrogen spectrum detected by the
PyroDetect system was evaluated against a set of substances.
Lipopolysaccharide is detected by the system down to
a concentration of 25 pg/ml, corresponding to 0.25 endotoxin units (EU). This reaction is enhanced by soluble
peptidoglycan (PGN) up to a concentration of 10 µg/ml,
resulting in a reduction of the limit of detection for LPS by
a factor of 10. In addition, a general increase in IL-1ß production was observed for all LPS concentrations.
Although this enhancing effect could increase sensitivity,
it may create a problem for pyrogen analyses according to
the European Pharmacopoeia. If the values for recovery of
a defined amount of control endotoxin (spike) exceed the
limits of 50 to 200 percent, the test is not regarded as valid
[ 4]. The interference may be overcome by dilution, but is
restricted by the contaminant limit concentration (CLC; for
intravenous infusions 5 EU/kg body weight/hour) and the
limit of detection (LOD) of the test system.
The MAT therefore may not be applicable for substances administered in large volumes. Alternatively, the
interfering effect offers the chance for a better differentiation of the inflammatory reactions. In the example of peptidoglycan, the pure substance alone does not induce any
IL-1ß response at concentrations between 10 pg/ml and
100 µg/ml (data not shown).
The pyrogenic activity of lipoteichoic acid could be de-
tected down to a concentration of less than 1 µg/ml. The
addition of peptidoglycan resulted in similar effects as
observed in the reaction to LPS, i.e. a marked reduction of
Pyrogenic activation by RNA reported for human TLR8
[ 5] was confirmed for E. coli RNA as well as synthetic
GU-rich 20-mer RNA molecules. As expected, no activation was induced by its counterpart with all uridin residues replaced by adenosin. For E. coli RNA, an LOD of
12. 5 µg/ml was calculated; the respective value for the
synthetic oligonucleotide was about a factor of 10 lower.
The MAT also detects the pyrogenic effect of poly (dT)
and its enhancement by the imidazoquinoline compound
R848, reflecting the cooperation between the receptors
TLR7 and TLR8 [ 6].
Results demonstrate that MAT is suitable for the detection of various pyrogenic substances which have a direct
or indirect influence on fever response in humans. In comparison to the RPT, the sensitivity of the MAT is equal or
better [ 7] but the MAT allows improved standardization
without requiring animal sacrifice.
The advantage of the MAT is even more pronounced
in comparison to the LAL test which only identifies
endotoxins and is subject to false positive reactions to
glucans [ 8].
Detection of the broad spectrum of pyrogens in pharmaceutical products using the MAT allows improved patient safety and offers the chance to abandon animal tests
for pyrogen control.
The data presented in this article was originally published in BIOspektrum (2010; 16, 779-781).
Download the application note with full results and data
at: www.merckmillipore.com/INTL/en/20140918_142235 n
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International validation of novel pyrogen tests based on human monocytoid cells. J Immunol Meth 298: 161-173
3. Schindler S, Spreitzer I, Löschner B. et al. (2006) International
validation of pyrogen tests based on cryopreserved human
primary blood cells. J Immunol Meth316: 42-51
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2. 6. 30 Monocyte Activation Test
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