Calprotectin - Bühlmann Laboratories

TM
BÜHLMANN fCAL
ELISA
Calprotectin
EK-CAL
96 tests
EK-CAL2
192 tests
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Unopened Reagents
INTENDED USE
TM
The BÜHLMANN fCAL ELISA kit is designed for the
extraction and quantitative determination of human
Calprotectin (MRP8/14; S100A8/S100A9) in stool samples
(ref. 1-3).
PRINCIPLE OF THE ASSAY
The test allows for the selective measurement of Calprotectinantigen by sandwich ELISA. A monoclonal capture antibody
(mAb) highly specific to the Calprotectin heterodimeric and
polymeric complexes (ref. 4-5), respectively, is coated onto
the microtiter plate. Calibrators, controls and patients extracts
are incubated at room temperature for 30 minutes. After a
washing step a detection antibody (Ab) conjugated to
horseradish peroxidase (HRP) detects the calprotectin
molecules bound to the monoclonal antibody coated onto the
plate. After incubation and a further washing step,
tetramethylbenzidine (TMB) will be added (blue color
formation) followed by a stopping reaction (change to yellow
color). The absorption is measured at 450 nm.
REAGENTS SUPPLIED AND PREPARATION
Reagents
EK-CAL
EK-CAL2
Quantity
3 bottles
6 bottles
Extraction Buffer
125 mL
x 125 mL
Microtiter Plate
precoated with
12 x 8
2 x 12 x 8
anti-Calprotectin
wells
wells
mAb
Plate Sealer
3 pieces
6 pieces
Wash Buffer
Concentrate
1 bottle
2 bottles
(10x) with
x 100 mL x 100 mL
preservatives
Incubation Buffer 2 bottles
3 bottles
with preservatives x 125 mL x 125 mL
Calibrators
A to E1) 2)
5 vials
5 vials
Calprotectin in a
x 1 mL
x 1 mL
buffer matrix with
preservatives
Control Low /
High3)
2 vials
2 vials
human serum with x 1 mL
x 1 mL
preservatives
Enzyme Label
Anti-Calprotectin
1 vial
2 vials
Ab conjugated to
x 12 mL
x 12 mL
HRP
TMB Substrate
1 vial
2 vials
TMB in citrate
x 12 mL
x 12 mL
buffer
Stop Solution
1 vial
2 vials
0.25 M sulfuric
x 12 mL
x 12 mL
acid
Code
2)
3)
B-CAL-EX
Ready to use
B-CAL-MP
Ready to use
2014-08-14
Extraction Buffer
Store at 2-8°C until expiration d ate.
Microtiter Plate
Return unused strips immediately to the foil pouch
containing the desiccant packs and reseal along the
entire edge of zip-seal.
Store until expiration date at 2-8°C.
Diluted Wash Buffer
Store for up to 6 months at 2-8°C.
Incubation Buffer
Calibrators
Controls
Store at 2-8°C until expiration date.
Enzyme Label
TMB-Substrate
Stop Solution
Table 2
REAGENTS SUPPLIED SUPPLEMENTARY
Fecal Extraction Devices
CALEX® Cap
Device
Package with 50 or 200
tubes respectively, each
filled with extraction buffer,
5 ml / ready to use
B-CALEX-C50
B-CALEX-C200
Smart-Prep
50 tubes, spatulas, and base
caps
B-CAL-RD
50 tubes consisting of tube,
cone & dosing tip,
1.3 mL, ready to use
B-CAL-SO50
Table 3
B-CAL-WB
Dilute each with
900 mL of
deionized H2O
B-CAL-IB
Ready to use
B-CALCASET
Ready to use
B-CALCONSET
Ready to use
B-CAL-EL
Ready to use
B-TMB12
Ready to use
B-STS12
Ready to use
Corrosive agent
The actual Calprotectin concentration of the standards A to E are 4, 12,
40, 120 and 240 ng/mL, respectively. For extraction and subsequent
sample dilution, a dilution of 1:2500 was taken into account for the
assignment of calibrator A to E. The following concentrations have to be
used for the lower range ELISA procedure: 10, 30, 100, 300 and 600
µg/g Calprotectin.
If you choose the extended range ELISA procedure the following
calibrator concentrations have to be used in the respective ELISA
protocol: 30, 90, 300, 900 and 1800 µg/g Calprotectin.
The controls contain lot specific amounts of native human Calprotectin.
Refer to the additional QC data sheet for actual concentrations.
Opened / Reconstituted Reagents
Table 1
1)
Store at 2-8°C. Do not use past kit expiration date printed on the labels.
2/36
MATERIALS REQUIRED BUT NOT PROVIDED
Extraction Procedure
• 10 µL disposable inoculation loops
• 15 mL polypropylene tubes with screw caps required for
standard extraction procedure; extraction devices (see
above).
• Laminar flow work station
• Multi tube vortex mixer
• Precision balance (10-150 mg)
• Micro centrifuge (≥3000 g)
• Centrifuge ((≥500 g)
ELISA Procedure
• 10, 100 and 1000 µL precision pipettes with disposable tips.
• Disposable polystyrene or polypropylene tubes for the
preparation of sample dilutions.
• 1000 mL cylinder to dilute the Wash Buffer.
• Microtiter plate washer (see Technical Precautions) or
squeeze bottle for Wash Buffer.
• Microtiter plate rotator (see Technical Precautions).
• Blotting paper.
• Microtiter plate reader for measurement of absorbance at
450 nm.
PRECAUTIONS
SAFETY PRECAUTIONS
• The microtiter-plate, calibrators and controls of this test
contain components of human origin. Although tested and
found negative for HBV surface antigen, HCV and HIV1/2
antibodies, the reagents should be handled as if capable
of transmitting infections and should be handled in
accordance with Good Laboratory Practices (GLP) using
appropriate precautions.
BÜHLMANN fCALTM ELISA
• Substrate and Stop Solution: The Substrate TMB (BTMB12) contains Tetramethylbenzidine, and hydrogen
peroxide (H2O2). The Stop Solution (B-STS12) contains
sulfuric acid (0.25 M). Each of those reagents is irritant to
eyes, skin and mucous membranes. Avoid contact with
Eyes, skin and cloths. After contact with eyes or skin,
wash immediately with plenty of water.
• Unused solution should be disposed of according to local
State and Federal regulations.
TECHNICAL PRECAUTIONS
Kit components
• Residues in the microtiter plate wells result from the
production process. They are removed in the washing
step (Assay procedure step 3) and do not affect the
results.
• Read carefully the instructions prior to carrying out the
test. Test performance will be adversely affected, if
reagents are incorrectly diluted, modified or stored under
conditions other than those as detailed in this instruction
for use.
• Components must not be used after the expiry date
printed on the labels.
• Do not mix different lots of reagents.
• Every effort should be made to ensure that no cross
contamination occurs between reagents, samples or
between wells.
• Let the reagents adjust to reach room temperature. Mix
well (vortex) the reagents before use.
• Microwells cannot be re-used.
Extraction:
• To receive quantitative results it is important to
homogenize the stool sample completely with the
extraction buffer. Avoid contamination at the top of the tube
- insoluble (undigested) components can still be in the tube
after extraction.
ELISA Procedure:
• In the ELISA procedure the washing step is essential to
guarantee reproducible results. A minimal incubation
time of the Wash Buffer in the wells of at least 20
seconds must be ensured each time.
• When using automated washer, BÜHLMANN strongly
recommends using “plate mode” i.e. each process step
(dispense/aspiration) is performed on all of the strips
sequentially, before processing to the next process step.
Thus, the minimal incubation time is guaranteed.
• The indicated no. of washing cycles is mandatory to
ensure reproducible results.
• Set the Plate rotator (shaker) at 450 rpm (<10 Hz). Higher
rotation frequency may cause poor dilution linearity at
values between 300/900 and 600/1800 µg/g. Orbital
rotation instead of reciprocal shaking should be used.
• To ensure a complete antigen/antibody interaction, the
incubation time in step 5 must be at least 30 minutes.
Moderately longer incubation time (up to 5 minutes ) has
no influence to the final outcome.
• The Enzyme Label is inactivated by oxygen and is highly
sensitive to sodium azide, thimerosal, hypochlorous acid
and aromatic chlorohydrocarbons often found in
laboratory water supplies. Therefore, use only deionized
high quality water.
• A new standard curve must be generated each time the
assay is performed. Vertical alignment is recommended.
• If the initial concentration of an unknown sample reads
above the concentration of the highest Calibrator,
Revision date:
2014-08-14
3/36
Calibrator E, the sample must be further diluted with
Incubation Buffer and assayed again according to the
assay procedure. The resulting total dilution factor must
be taken into account for the calculation of results.
SPECIMEN COLLECTION AND STORAGE
If the extraction devices are used less than 1 g of native stool
sample is needed for the extraction procedure.
Collect stool samples into plain tubes. The samples can be
stored refrigerated at 2-8°C for at least 6 days.
The extracts are stable for at least 7 days at 2-8ºC and for at
least 24 months at -20ºC.
Important: The sample must be collected without any
chemical or biological additions in the collection device.
STANDARDIZATION
TM
The test BÜHLMANN fCAL ELISA is calibrated against
purified MRP8/14 from human granulocytes.
STOOL SAMPLE EXTRACTION
Standard extraction procedure
1. Label and weigh (tare) the empty polypropylene tube
together with the inoculation loop.
2. Take out 50 to 100 mg of the stool sample by means of the
inoculation loop and place it into the pre-weighted tube.
3. Calculate the net amount of sample, break off the
inoculation loop and leave the lower part of the loop in the
tube.
4. Add Extraction Buffer according to the formula
x mg stool x 49 = y µl extraction buffer
(e.g. 50 mg stool + 2450 µl buffer) to the tube and close
the tube
5. Homogenize the sample on a multi tube vortexer by
vigorous shaking (at highest speed) for 30 minutes.
6. Transfer the homogenate into a 2 mL Eppendorf tube and
centrifuge in a microcentrifuge for 5 minutes at 3’000 x g.
7. Take the supernatant into a fresh, labeled tube and
continue with the ELISA procedure.
Extraction procedures using fecal extraction devices:
The extraction procedure is described and illustrated in the
instruction for use delivered with the respective extraction
device.
®
1. CALEX Cap Device (Code B-CALEX-C50/B-CALEXC200): The extraction tubes are prefilled with extraction
buffer.
®
Important: After extraction, centrifuge the CALEX Cap
Device for 5 minutes at 500 x g and continue with the ASSAY
PROCEDURE.
2. Fecal Extraction Device Roche (Code 10745804322) or
BÜHLMANN Smart-Prep (Code: B-CAL-RD).
®
TM
3. ScheBo
Quick-Prep
(Code B-CAL-SO50):
extraction tubes are prefilled with extraction buffer.
The
Important: After extraction with BÜHLMANN Smart Prep and
®
TM
ScheBo Quick-Prep , centrifuge the tubes for 5 minutes at
3’000 x g. Alternatively, transfer the homogenate into a 2 mL
Eppendorf tube and centrifuge it in a microcentrifuge for 5
minutes at 3’000 x g. Decant the supernatant into a fresh,
labeled tube and continue with the ASSAY PROCEDURE.
BÜHLMANN fCALTM ELISA
WORKING RANGE
The assay can be performed according to the following
procedures – lower or extended range ELISA
procedure. Which procedure is to be chosen depends
on the expected Calprotectin concentration of the
samples. For samples up to 600 µg/g choose the lower
range procedure (working range 10 – 600 µg/g)). If the
samples tend to exceed 600 µg/g choose the extended
range procedure (working range 30 – 1800 µg/g).
ASSAY PROCEDURE
Important:
Allow the reagents to equilibrate to 18-28 °C prior to use.
Only dilute stool extracts. Standards and controls are ready
to use.
1. SAMPLE DILUTION OPTIONS
WORKING RANGE 10 – 600 µg/g
1.1. Manual weighing procedure, Smart Prep, or
®
TM
ScheBo Quick-Prep : Dilute the stool extracts 1:50
with Incubation Buffer (e.g. 20 µL extract and 980 µL
incubation buffer) and mix well. Let the samples
equilibrate for at least 5 minutes at 18-28°C prior to
proceeding to step 4c.
®
1.2. CALEX Cap Device: Dilute the stool extracts 1:5
with Incubation Buffer (e.g. 100 µL extract and 400 µL
incubation buffer) and mix well. Let the samples
equilibrate for at least 5 minutes at 18-28°C prior to
proceeding to step 4c.
WORKING RANGE 30 – 1800 µg/g
The working range can be extended by a factor of 3, if
you dilute the samples 1:7500 instead of 1:2500. This
procedure is recommended, if high Calprotectin
concentrations are to be expected. Precision and
linearity of the assay allow for this extension of the
measuring range.
1.1. Manual weighing procedure, Smart Prep, or
®
TM
ScheBo Quick Prep : Dilute the stool extracts
1:150 with Incubation Buffer (e.g. 20 µL extract and
2980 µL incubation buffer) and mix well. Let the
samples equilibrate for at least 5 minutes at 18-28°C
prior to proceeding to step 4c.
®
1.2. CALEX Cap Device: Dilute the stool extracts 1:15
with Incubation Buffer (e.g. 50 µL extract and 700 µL
incubation buffer) and mix well. Let the samples
equilibrate for at least 5 minutes at 18-28°C prior to
proceeding to step 4c.
2. Prepare a plate with sufficient strips to test the required
number of calibrators, controls and diluted samples.
Remove excess strips from the holder and re-seal them
in the foil pouch together with the desiccant packs
without delay. Store refrigerated.
3. Wash the coated wells twice using at least 300 µL of
Wash Buffer per well. Empty the wells and tap the plate
firmly onto blotting paper.
Important: For every of the three wash steps a minimal
incubation time of at least 20 seconds of the Wash Buffer in
the wells must be ensured (see Technical Precautions –
ELISA Procedure).
4a. Pipet 100 µL of Incubation Buffer (Blank) and
Pipet 100 µL of Calibrator A-E into the respective wells.
Revision date:
2014-08-14
4/36
4b. Pipet 100 µL of the Low and High Controls into the
respective wells.
4c. Pipet 100 µL of each diluted sample into the subsequent
wells.
5. Cover the plate with a plate sealer, and incubate for
30 + max. 5 min on a plate rotator set at 450 rpm at 1828 °C (see Technical Precautions – ELISA Procedure) .
6. Remove and discard the plate sealer. Empty the wells
and wash three times using at least 300 µL of Wash
Buffer per well (see Technical Precautions – ELISA
Procedure). Empty the wells and tap the plate firmly
onto blotting paper.
7. Pipet 100 µL of Enzyme Label to each well.
8. Cover the plate with a plate sealer, and incubate for 30
± 5 min on a plate rotator set at 450 rpm at 18-28 C.
9. Remove and discard the Plate Sealer. Empty the wells
and wash five times using at least 300 µL of Wash
Buffer per well. Empty the wells and tap the plate firmly
onto blotting paper.
Important: Allow the TMB Substrate Solution to equilibrate to
18-28 C.
10. Pipet 100 µL of the TMB Substrate Solution to all wells.
11. Cover the plate with a plate sealer, protect the plate
from direct light and incubate for 15 ± 2 min on a plate
rotator set at 450 rpm at 18-28°C.
12. Pipet 100 µL of Stop Solution to all wells. Remove air
bubbles with a pipette tip. Proceed to step 13 within 30
min.
13. Read the absorbance at 450 nm in a microtiter plate reader.
RESULTS & CALCULATION
Read the absorbance at 450 nm in a microplate reader for
each calibrator, control and sample using a 4 PL fit with
blank subtraction and have the concentration of the samples
calculated.
WORKING RANGE 10 – 600 µg/g
If you choose the lower range ELISA procedure, the
following calibrator concentrations have to be used in the
respective ELISA protocol: 10, 30, 100, 300 and 600 µg/g
Calprotectin.
Additional dilution factors have to be multiplied with the
results to obtain the final results.
Refer to Table 19 and Figure 1 for typical data (results and
standard curve). These results and standard curve are
provided for demonstration purposes only. A standard curve
must be generated for each set of samples to be assayed.
WORKING RANGE 30 – 1800 µg/g
If you choose the extended range ELISA procedure, the
following calibrator concentrations have to be used in the
respective ELISA protocol: 30, 90, 300, 900 and 1800 µg/g
Calprotectin.
Additional dilution factors have to be multiplied with the
results to obtain the final results.
Refer to Table 24 and Figure 3 for typical data (results and
standard curve). These results and standard curve are
provided for demonstration purposes only. A standard
curve must be generated for each set of samples to be
assayed.
BÜHLMANN fCALTM ELISA
PERFORMANCE CHARACTERISTICS
WORKING RANGE: 10 – 600 µg/g
Assay performance characteristics have been established
in duplicates.
Intra-Assay Precision: 4.7 %. The intra-assay precision was
calculated from 20 pairs of values from 3 extracted stool
samples assayed in a single run according to the assay
procedure. The values are presented in Table 20.
Inter-Assay Precision: <15 %. The inter-assay precision of
the ELISA was calculated from 5 extracted stool samples. The
aliquots were tested according to the assay procedure in 10
different runs by three technicians using 2 kit lots in two
different labs. The values are presented in Table 21.
Detection limit (LoB): <10 µg/g. Twenty duplicates of
Incubation Buffer were assayed in a single run. Mean and
standard deviation were calculated for the absorbance values.
The minimal detectable dose of Calprotectin was calculated to
be clearly below Calibrator A (10 µg/g) by adding two
standard deviations to the mean absorbance and intersecting
this value with the standard curve obtained in a new run.
Detection limit (LoQ): <10 µg/g. Ten stool samples with
values between 5.2 and 1254 µg/g Calprotectin were assayed
20 times in duplicates in one assay. The %CV and the mean
values were calculated for each sample. The functional
sensitivity was observed at 15 % CV. The resulting precision
profile (Figure 2) allows the precise measurement within the
whole standard range from 10 to 600 µg/g.
Dilution Linearity: 103 %. Seven stool samples with
elevated Calprotectin values were extracted according to the
assay procedure. The extracts were diluted with Incubation
Buffer and subsequently assayed according to the assay
procedure. The expected values were calculated from the
observed value found with the first dilution. The results are
presented in Table 22.
Spiking Recovery: 100 %. Two extracted stool samples
were spiked with different amounts of diluted, Calprotectin
containing human serum. The samples were measured
before and after spiking according the assay procedure. The
results are presented in
Table 23.
Crossreactivity: <0.1 %. Incubation Buffer spiked with
different amounts of recombinant MRP8 and MRP14 were
measured according to the assay procedure. The values are
presented in Table 28
WORKING RANGE: 30 – 1800 µg/g
Intra-Assay Precision: 4.0 %. The intra-assay precision
(mean) was calculated from the results of 20 duplicates from 3
extracted stool samples assayed in a single run according to
the assay procedure. The values are presented in Table 25.
Inter-Assay Precision: <15 %. The inter-assay precision of
the ELISA was calculated from 5 extracted stool samples. The
aliquots were tested according to the assay procedure in 10
different runs by three technicians using 2 kit lots in two
different labs. The values are presented in Table 26.
Detection limit (LoQ): <30 µg/g. 18 stool samples with
values between 10.8 and 2080 µg/g Calprotectin were
measured 20 times in one assay. The % CV and the mean
values were calculated for each sample. The LoQ was
observed at 15 % CV. The resulting precision profile allows
the precise measurement within the whole standard range
from 30 to 1800 µg/g. The results are presented in Figure 4.
Dilution Linearity: 102 %. Five stool samples with elevated
Calprotectin concentrations were extracted according to the
assay procedure. The extracts were diluted with Incubation
Buffer and subsequently assayed according to the assay
procedure. The expected values were calculated from the
Revision date:
2014-08-14
5/36
observed value found with the first dilution. The results are
presented in Table 27.
INTERPRETATION OF RESULTS
Estimation of faecal Calprotectin is a reliable and easy way
to distinguish organic from functional gastrointestinal
diseases.
In a clinical study 401 symptomatic patients scheduled for
colonoscopy were investigated. Endoscopy examination
showed 273 patients with functional diseases whereas 128
patients had various organic diseases (colitis, Crohn’s,
ulcers, diverticulitis, polyps, adenomas, cancer, or
infectious diseases) (ref.12).
ROC curve analysis (AUC: 0.935) resulted in an optimal
clinical cut-off at 50 µg/g. Applying this cut-off, a clinical
sensitivity and specificity of 84.4% and 94.5%, respectively
can be reached in the differentiation between organic and
functional diseases (see Table 29).
Faecal Calprotectin levels from adults and children are
comparable, whereas levels of newborns can be
significantly increased (ref. 8).
These data support the following recommendation for
interpretation of results:
Normal values below 50 µg/g:
Calprotectin values <50 µg/g are not indicative of
inflammation in the gastrointestinal tract. Patients with low
Calprotectin levels are likely not to be in need of invasive
procedures to determine the inflammation cause (ref. 12).
Elevated values between 50 and 200 µg/g:
Calprotectin values between 50 and 200 µg/g can
represent mild organic disease such as inflammation
caused by NSAIDs, mild diverticulitis and IBD in remission
phase. The low inflammatory response shown within this
range may suggest repeating the measurement and
performing further investigations.
Elevated values above 200 µg/g:
Calprotectin values >200 µg/g are indicative of active
organic disease with inflammation in the gastrointestinal
tract. Appropriate further investigative and curative
procedures by specialists are suggested.
The cut-off level suggested for adults (50 µg/g) can also be
used for children aged from 4 to 17 years regardless of sex
(ref. 9).
QUALITY CONTROL
Thorough understanding of this instruction is necessary for
the successful use of the product. Reliable results will be
obtained only by precise laboratory techniques (current
GLP guidelines) and accurately following this instruction for
use.
Since there is no control for Calprotectin commercially
available, we recommend using a pool of positive stool
extractions for internal quality control.
The reproducibility of standard curve parameters and control
values should be within established limits of laboratory
acceptability. The confidence limits for the Controls are lotspecific and printed on the additional QC data sheet.
If the precision of the assay does not meet the established
limits and repetition has excluded errors in technique, check
the following issues: i) pipetting, temperature controlling and
timing devices ii) ELISA reader settings iii) expiration dates
of reagents iv) storage and incubation conditions v) TMB
Substrate Solution should be colorless vi) purity of water.
BÜHLMANN fCALTM ELISA
PERFORMANCE LIMITATIONS
• Reagents delivered with this kit are being optimized for
the determination of Calprotectin from human stool
samples.
• Test results should be interpreted in conjunction with
information available from clinical assessment of the
patient and other diagnostic procedures.
EK-CAL
EK-CAL2
Rekonstitution
B-CAL-EX
8 x 12
Kavitäten
2 x 8 x 12
Kavitäten
B-CAL-MP
3 Stück
B-CAL-WB
B-CAL-IB
B-CALCASET
B-CALCONSET
B-CAL-EL
B-TMB12
B-STS12
2014-08-14
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BÜHLMANN fCALTM ELISA
1)
2)
3)
Stopp-Lösung
Table 5
B-CALEX-C50
B-CALEX-C200
Smart-Prep
B-CAL-RD
B-CAL-SO50
2014-08-14
TMB-Substrat
7/36
2014-08-14
8/36
BÜHLMANN fCALTM ELISA
1.2. CALEX
Cap Device: Die Stuhlextrakte mit
Inkubationspuffer 1:5 verdünnen (z.B. 100 µL Extrakt +
400 µL Inkubationspuffer). Nach der Verdünnung gut
mischen und die Proben vor Gebrauch in Schritt 4c
mindestens 5 Minuten bei 18-28°C stehen lassen.
MESSBEREICH 30 – 1800 µg/g
The working range can be extended by a factor of 3, if
you dilute the samples 1:7500 instead of 1:2500. This
procedure is recommended, if high Calprotectin
concentrations are to be expected. Precision and
linearity of the assay allow for this extension of the
measuring range.
®
3.
2014-08-14
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BÜHLMANN fCALTM ELISA
Die Leistungsmerkmale
evaluiert.
2014-08-14
10/36
EK-CAL
EK-CAL2
Code
2014-08-14
11/36
B-CALMP
B-CALWB
2 flacons de 1
mL
BÜHLMANN fCALTM ELISA
1)
2)
3)
Table 8
B-CALEX-C50
Smart-Prep
B-CAL-RD
B-CALEX-C200
B-CAL-SO50
Table 9
2014-08-14
12/36
2014-08-14
13/36
TM
BÜHLMANN fCALTM ELISA
3.
6.
7.
8.
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BÜHLMANN fCALTM ELISA
2014-08-14
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EK-CAL
EK-CAL2
2014-08-14
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3 fogli
6 fogli
B-CAL-IB
5 provetteda B-CAL1 mL
CASET
1 provetta
da 12 mL
B-TMB12
B-STS12
BÜHLMANN fCALTM ELISA
2)
3)
PRECAUZIONI
PRECAUZIONI DI SICUREZZA
PRECAUZIONI TECNICHE
CALEX Cap
Smart-Prep
ScheBo®
TM
Quick-Prep
B-CALEX-C50
B-CALEX-C200
B-CAL-RD
B-CAL-SOFI
Table 12
2014-08-14
17/36
TM
TM
2014-08-14
18/36
6.
7.
8.
9.
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RANGE DI MISURAZIONE: 30 – 1800 µg/g
2014-08-14
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BÜHLMANN fCALTM ELISA
ALMACENAMIENTO Y DURACIÓN DE LOS REACTIVOS
TM
EK-CAL
EK-CAL2
Reconstituci
ón
B-CALEX-C50
B-CALEX-C200
B-CAL-EX
Listo para
usar
Smart-Prep
B-CAL-RD
B- CAL MP
B-CAL-SO50
B- CAL WB
B-CALCASET
Listo para
usar
2 viales
de 1 mL
B-CALCONSET
Listo para
usar
2 viales
de 12 mL
B- CAL -EL
Listo para
usar
2 viales
de 12 mL
B-TMB12
2014-08-14
Table 15
PRECAUCIONES
REACTIVOS INCLUIDOS Y PREPARACIÓN
Reactivos
21/36
2014-08-14
22/36
TM
®
®
6.
7.
8.
9.
2014-08-14
23/36
2014-08-14
24/36
2014-08-14
25/36
1)
2)
3)
EK-CAL
EK-CAL2
3 peças
6 peças
B-CAL-MP
B-CALCASET
B-CALCONSET
B-CAL-EL
B-TMB12
Pronto para
uso
B-STS12
Device
Smart-Prep
B-CAL-RD
B-CAL-SO50
CALEX® Cap
B-CALEX-C200
Table 17
2014-08-14
26/36
TM
®
2014-08-14
27/36
3.
6.
7.
8.
9.
2014-08-14
5.
28/36
do
teste
foram
BÜHLMANN fCALTM ELISA
2014-08-14
29/36
BÜHLMANN fCALTM ELISA
APPENDIX I
REFERENCES/ LITERATURREFERENZEN/ RÉFÉRENCES/ RIFERIMENTI/ REFERENCIAS
1. Striz I and Trebichavsky I: Calprotectin – a
pleiotropic molecule in acute and chronic
inflammation. Physiol Res. 53, 245-253 (2004)
2. Tibble JA et al.: Use of surrogate markers of
inflammation and Rome criteria to distinguish
organic from nonorganic intestinal disease.
Gastroenterol 123, 450-460 (2002)
3. Fagerhol MK: Calprotectin, a faecal marker of
organic gastrointestinal abnormality. Lancet 356,
1783-4 (2000)
4. Hessian PA and Fisher L: The heterodimeric
complex of MRP-8 (S100A8) and MRP-14
(S100A9). Antibody recognition, epitope definition
and the implications for structure. Eur J Biochem
268, 353-63 (2001).
5. Goebeler M et al.: Expression and complex
formation of S100-like proteins MRP8 and MRP14
by macrophages during renal allograft rejection.
Transplantation 58, 355-61 (1994).
6. Tibble JA et al.: A simple method for assessing
intestinal inflammation in Crohn’s disease. Gut 47,
506-513 (2000).
7. Wassell J et al.: Faecal Calprotectin: a new marker
for Crohn’s disease? Ann Clin Biochem 41, 230232 (2004)
2014-08-14
8. Konikoff MR and Denson LA: Role of fecal
calprotec-tin as a biomarker of intestinal
inflammation in inflam-matory bowel disease.
Inflamm Bowel Dis 12(6), 524-34 (2006)
9. Fagerberg UL et al.: Colorectal inflammation is well
predicted by fecal calprotectin in children with
gastrointestinal symptoms. J Pediatr Gastroenterol
Nutr 40, 450-5 (2005)
10. Johnson M W et al.: Faecal calprotectin: a
noninvasivee diagnostic tool and marker of severity
in pouchitis. Eur J Gastroenterol Hepatol 20 :174179, 2008)
11. Sipponen T et al. Faecal Calprotectin, Lactoferrin,
and Endoscopic Disease Activity in Monitoring AntiTNF-alpha Therapy for Crohn’s Disease. Aliment
Pharmacol Ther 28, 1221–1229, (2008)
12. Manz, M. et al. Value of fecal calprotectin in the
evaluation of patients with abdominal discomfort:
an observational study. BMC Gastroenterology 12,
5 (2012).
13. Jahnsen J, Røseth AG, Aadland E. Measurement
of calprotectin in faeces.. Tidsskr Nor Legeforen
128, 743–5 (2008)
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BÜHLMANN fCALTM ELISA
APPENDIX II
TABLES/ TABELLEN/ TABLES/ TABELLE/ TABLAS
LOWER RANGE PROCEDURE 10 - 600 µg/g
Table 19:
Example of Results
Conc.
[µg/g]
Blank Avg.
Cal A
Cal A
Cal A Avg.
Cal B
Cal B
Cal B Avg.
Cal C
Cal C
Cal C Avg.
Cal D
Cal D
Cal D Avg.
Cal E
Cal E
Cal E Avg.
Ctrl Low
Ctrl Low
Ctrl Low Avg.
Ctrl High
Ctrl High
Ctrl High Avg.
low
medium
high
Standard_Curve
0.073
0.066
0.069
0.143
0.153
0.148
0.465
0.456
0.460
1.121
1.135
1.128
1.658
1.671
1.664
0.201
0.189
0.195
0.598
0.583
0.590
2
7.2
1.5
4.8
1
1.4
0.5
0.9
0
10
4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D:
0.6
41
39
40
134
130
132
STD_CAL (Standards: Concentration v s OD)
__________
CV
[%]
1.4
5.6
33.0
2.7
3.2
8.1
C
D
R^2
400
2.67
1
1:200
1:400
1:800
1:1600
1:3200
1:6400
1:12800
Mean
1:50
1:100
1:200
1:400
1:800
Mean
1:400
1:800
1:1600
1:3200
1:6400
Mean
Mean
Mean
Mean
Mean
S1
Inter-Assay Precision
SD
[µg/g]
2.5
6.4
7.9
15.0
57.8
CV
[%]
13.5
14.5
10.7
6.6
11.1
11.3
S2
Precision Profile
S4
S5
S6
S7
Mean
60
40
Dilution Linearity/Parallelism
Observed
[µg/g]
405
182
95
49
25
15.6
6.6
Dilution
S3
Intra Assay CV [%CV] n=20
B
1.19
1.8
SD
[µg/g]
A
0.0354
4.4
4.7
Mean
[µg/g]
18.1
44.5
74.3
227
520
1000
Curv e Fit Option - Fixed Weight Value
Mean
Table 21:
100
Concentration (µg/g)
Intra-Assay Precision
Mean
[µg/g]
52.5
173.8
408.5
Example of a Standard Curve
CV Conc
[%]
0.096
10
10
10
30
30
30
100
100
100
300
300
300
600
600
600
Calc. Conc.
[µg/g]
Expected
[µg/g]
202
101
51
25
12.7
6.3
232
124
61
28
12
116
58
29
15
290
145
73
39
19
145
72
36
18
15 % CV
Table 23:
20
Spiking Recovery
µg/g
0
1
10
100
1000
Calprotectin [µg/g]
10000
S1
5.3
spiked with
[µg/g]
10
30
100
150
300
400
Observed
[µg/g]
13.6
33.6
101
147
287
468
Expected
[µg/g]
15.3
35.3
105
155
305
405
Mean
10
600
S2
24.6
Mean
Mean
2014-08-14
31/36
10
30
100
150
300
400
32.9
49.2
139
176
358
467
34.6
54.6
125
175
325
425
BÜHLMANN fCALTM ELISA
APPENDIX III
TABLES/ TABELLEN/ TABLES/ TABELLE/ TABLAS
EXTENDED RANGE PROCEDURE 30-1800 µg//g
Table 24:
Example of Results
Conc.
[µg/g]
30
30
30
90
90
90
300
300
300
900
900
900
1800
1800
1800
0.057
0.047
0.046
0.047
0.138
0.140
0.139
0.464
0.452
0.458
1.207
1.192
1.200
1.627
1.630
1.629
0.147
0.162
0.155
0.618
0.618
0.618
Calc. Conc.
[µg/g]
Standard_Curve
0.9
1.5
1.0
1
1.9
37.7
713
1246
0.8
0
10
0.1
105
115
110
396
396
396
6.2
SD
[µg/g]
CV
[%]
2.5
20.1
32.4
6.7
2.8
2.6
SD
[µg/g]
9.7
20
62
111
221
B
1.47
C
736
D
2.06
R^2
1
Precision Profile
80
70
60
50
40
<15 % CV
30
20
10
CV
[%]
12.8
8.8
7.8
11.1
12.6
10.6
0
1.0
10.0
100.0
1000.0
10000.0
Calprotectin [ µg/g]
30
S1
S2
S3
S4
S5
Mean
2014-06-30
10000
0.6
Table 27:
1000
4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D:
A
STD_CAL (STD_CAL: Concentration vs Values)
0.0371
__________
Curve Fit Option - Fixed Weight Value
Inter-Assay Precision
Mean
[µg/g]
75.5
225
788
1000
1764
100
µg/g
4.0
Table 26:
0.5
Intra-Assay Precision
Mean
[µg/g]
Example of a Standard Curve
2
Mean
CV Conc
[%]
Intra Assay CV [%] n=20
Blank Avg.
Cal A
Cal A
Cal A Avg.
Cal B
Cal B
Cal B Avg.
Cal C
Cal C
Cal C Avg.
Cal D
Cal D
Cal D Avg.
Cal E
Cal E
Cal E Avg.
Ctrl Low
Ctrl Low
Ctrl Low Avg.
Ctrl High
Ctrl High
Ctrl High Avg.
32/36
1800
Dilution Linearity/Parallelism
Dilution
1:225
1:350
1:1200
1:2400
1:4800
Mean
1:100
1:120
1:150
1:200
1:400
1:800
Mean
Mean
Mean
Mean
Observed
[µg/g]
2466
1296
420
237
111
758
702
552
406
210
108
Expected
[µg/g]
1585
462
233
116
632
505
379
190
95
BÜHLMANN Calprotectin ELISA
Table 28*
Cross Reactivity
MRP8
[ng/mL]
Spiked with
100 µg/mL
10 µg/mL
1 µg/mL
100 ng/mL
10 ng/mL
26.0
8.0
<4.0
<4.0
<4.0
Table 29 (ref.12)
n
cut-off
Sensitivity
Specificity
PPV
NPV
LR+
LR-
MRP14
[ng/mL]
38.7
3.4
<4.0
<4.0
<4.0
Clinical Study
Calprotectin
(EK-CAL)
401
50 µg/g
84.4%
94.5%
87.8%
92.8%
15.4
8.25
2014-08-14
Lactoferrin
391
3 µg/mL
74.2%
91.0%
79.3%
88.4%
0.17
0.28
33/36
* Data have been established with the lower range ELISA
procedure
Table
description:
cf.
“Results”,
“Performance
Characteristics” and “Interpretation of Results”.
Tabellenbeschreibung: siehe Resultate
“Leistungsmerkmale” und “Interpretation der Resultate”.
Explications aux tableaux: voir “Résultats”,
“Caracteristiques de Performance” et “Interprétation des
Résultats”.
Descrizione tavola: cf. “Risultati”, “Caratteristiche di
Prestazione” e “interpretazione dei resultati”.
Explicaciones a las Tablas: ver “Resultados”,
“Características de Efficiencia”) y “Interpetaciones de los
resultados.”
Explicação a Tablas: ver “Resultados”, “Características
de desempenho” y “Interpretação do resultados
BÜHLMANN fCALTM ELISA
APPENDIX IV
SHORT PROTOCOL
CALPROTECTIN EXTRACTION
Standard Extraction Procedure
0.0 mg
75.0 mg
Pre-weigh empty tube
+ Inoculation loop
Weigh 50 to100 mg
faeces
Transfer ~1.0 ml
into a fresh tube
Add 49 volumes
of 1x B-CAL-EX
Centrifuge 5 min
at 3’000 x g
Close tube and
vortex vigorously
for 30 min
Transfer supernatant into a fresh
tube and continue with the lower
or extended range ELISA
procedure (1:50 or 1:150).
CALPROTECTIN ELISA
Precoated Microtiter Plate
wash 2 x
100 µL Calibrators Controls
or diluted Samples
incubate 30 (+ 5) minutes
at 18-28°C on a plate rotator
wash 3 x
add 100 µL Enzyme Label
incubate 30 +/- 5 minutes
at 18-28°C on a plate rotator
wash 5 x
add 100 µL TMB Substrate
incubate 15 +/- 2 minutes
at 18-28°C on a plate rotator
add 100 µL Stop Solution
Read absorbance at 450 nm (within 30 minutes)
TIME TO RESULT: 75 MINUTES
2014-08-14
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BÜHLMANN fCALTM ELISA
NOTES
2014-08-14
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BÜHLMANN fCALTM ELISA
REF
LOT
IVD
MP
Explanation
Symbol
Use By
Verwendbar bis
Utiliser jusqu’au
Utilizzare entro
Fecha de caducidad
Data de expiração
Order Code
Bestellnummer
Code
Codice
Código
Código
Batch code
Chargenbezeichnung
Code du lot
Codice del lotto
Código de lote
Código lote
In Vitro Diagnostic Medical Device
In Vitro Diagnostikum
Dispositif médical de diagnostic in vitro
Dispositivo medico-diagnostico in vitro
Producto sanitario para diagnóstico in vitro
Producto sánitario para diagnóstico in vitro
Contains sufficient for <n> tests
Ausreichend für ”n” Ansätze
Contenu suffisant pour „n“ tests
Contenuto sufficiente per „n“ saggi
Contenudo sufficiente para <n> ensayos
Contenudo sufficiente para <n> tests
Consult Instructions for UseGebrauchsanweisung beachten
Consulter le mode d’emploi
Consultare le istruzioni per l‘uso
Consulte las instrucciones de uso
Leia cuidadosamente as instruções
Temperature limitation
Zulässiger Temperaturbereich
Limites de température
Limiti di temperatura
Limite de temperatura
Lίmite de temperatura
Extraction Buffer
Extraktions-Puffer
Tampon d’extraction
Tampone per estrazione
Tampón de extracción
Tampão extração
Microtiter plate
Mikrotiterplatte
Microplaque
Micropiastra
Microplaca
Microplaca
BUF WASH 10X
BUF INC
CAL A
-
CAL E
CONTROL H
EL
Explanation
Wash Buffer Concentrate (10x)
Wasch-Puffer Konzentrat (10x)
Concentré de tampon de lavage (10x)
Tampone di lavaggio concentrato (10x)
Tampón de lavado concentrado (x10)
Concentrado de tampão de lavagem
Incubation Buffer
Inkubations-Puffer
Tampon d’incubation
Tampone di incubazione
Tampón de incubación
Tampão de incubação
Calibrator A -E
Kalibrator A -E
Calibrateur A -E
Calibratore A - E
Calibrador A – E
Calibrador A – E
Control Low
Kontrolle tief
Contrôle bas
Controllo basso
Control bajo
Controle baixo
Control High
Kontrolle hoch
Contrôle élevé
Controllo alto
Control alto
Controle alto
Enzyme Label
Enzym-Marker
Marqueur enzymatique
Marcato enzimatico
Marcador enzimático
Marcador enzimático
TMB Substrate
TMB-Substrat
Substrat TMB
Substrato di TMB
Substrato de TMB
Substrato TMB
Stop Solution
Stopp-Lösung
Solution stop
Soluzione stoppante
Solución de parada
Solução stop
CALEX® is a registered trademark of BÜHLMANN in many countries of
the world.
Printing Date
2014-08-15
2014-08-14
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BÜHLMANN fCALTM ELISA