Internal Assay Control:
The preparation of an IQC material suitable
for monitoring the consistency of your assay, is
a slightly more involved process during which
you will need to make a bulk extract:
For this type of IQC, I would recommend
that you prepare three different extracts each
containing a different level of Calprotectin:
one at a measurable level below the cut-off
e.g. ~50 μg/g, one at or slightly above the
cut-off (~80-120 μg/g) and one somewhere
between 200-600 μg/g.
■ For each IQC level, prepare homogenised
stool using the method outlined above.
■ Next, decide on the total volume of extract
you wish to prepare. This will depend
upon how often you intend to run the IQC,
the volume required for each analysis
and the manufacturer’s quoted stability for
the extract when frozen. If it is not
possible to purchase the buffer directly from
the manufacturer, you may need to harvest
it from the extraction devices.
■ The kit insert for the extraction buffer/device
will quote the required ratio of stool to
buffer. For example, the CALEX® Cap
requires a stool:buffer ratio of 1:500 so if
you wish to make up 100 mL of extract,
weigh out 200 μg stool and add 100 mL
■ Using an accurate balance, weigh out the
correct mass of stool for the volume of
extract being prepared (see ratio quoted in
■Weigh the stool into the container in which
the extract will be made. For liquid stool
samples, use a pipette to measure out the
recommended volume of stool required.
■ Based on the mass of stool weighed into
the container, calculate the exact volume
of buffer required to give the correct ratio
of stool:buffer quoted in the kit insert.
■ Using the most accurate tool to hand
(volumetric pipette, measuring cylinder
etc.), measure out the total volume of
buffer needed and add it into the vessel
containing the stool.
■ Vortex the extract for 30 minutes.
Centrifuge the extract at 3000g for 10
minutes. Decant off the supernatant,
portion into single-use aliquots and freeze
at -80°C* for a minimum of 24 hours.1
Setting the Target Value
■ For each different level of IQC, thaw
several aliquots of extract (according to
■ Thoroughly mix each aliquot gently by
inversion, analyse each aliquot and take a
mean of the results obtained.
■ Set your acceptance criteria for the IQC
around this mean.
■ Each time you run your assay, remove an
aliquot of each different level of IQC from
the freezer, thaw, mix-well and analyse in
the usual way.
And there you have it, a simple IQC
preparation that is tailored for use with
*If you do not have access to a -80°C freezer,
store at the lowest temperature available and
follow the manufacturer’s guidance regarding
stability at that temperature. Do not analyse
stool or extract that has been stored for longer
than the manufacturer recommends.
1. Whitehead, S, French, J, Brookes, M et al.
Between-assay variability of faecal calprotectin
enzyme-linked immunosorbent assay kits.
Ann Clin Biochem 2013; 50: 53–61.
CALEX® Cap - Faecal Calprotectin Extraction Made Easy
Faecal calprotectin sample preparation for the
BÜHLMANN assays is quick, clean and consistent with
the CALEX® Cap extraction device.
It offers a simplified workflow solution for faecal
calprotectin extraction without the need to weigh,
decant or further dilute samples.
The tube is pre-filled with buffer to give optimal dilution
for maximum extraction efficiency.
CALEX tubes can be directly loaded onto automated
ELISA processors or laboratory track systems.
Find out more at www.calprotectin.co.uk/calex
Printed on Recycled Paper www.alphalabs.co.uk 9