ORGENTEC Diagnostika GmbH

PRINCIPLE OF THE TEST

Carl-Zeiss-Straße 49-51
Anti-Calprotectin antibodies are coated onto microwells.
55129 Mainz - Germany
Phone: +49 (0) 61 31 / 92 58-0 Calprotectin contained in a stool sample has to be released from the stool with extraction medium. Then, extracted
Fax: +49 (0) 61 31 / 92 58-58 Calprotectin can be analyzed with the Calprotectin ELISA assay.
Internet: www.orgentec.com The determination is based on an indirect enzyme linked immune reaction with the following steps:
Calprotectin extracted from the patient´s stool sample binds to the antibody coated on the surface of the reaction
wells. After incubation, a washing step removes unbound calprotectin. Subsequently added enzyme conjugate
i 580_4 binds to the immobilized antibody-antigen-complexes. After incubation, a second washing step removes unbound
enzyme conjugate. After addition of substrate solution the bound enzyme conjugate hydrolyses the substrate
forming a blue coloured product. Addition of an acid stopps the reaction generating a yellow end-product. The
intensity of the yellow color correlates with the concentration of the antibody-antigen-complex and can be measured
ORG 580 Calprotectin photometrically at 450 nm.

WARNINGS AND PRECAUTIONS

INTENDED PURPOSE • All reagents of this kit are intended for professional in vitro diagnostic use only.
Calprotectin is an ELISA-based test system for the quantitative measurement of calprotectin in human stool • The calibrators and controls contain calprotectin of human origin. Since no test can guarantee the absence of
samples to be used in the assessment of intestinal inflammatory disorders. This product is intended for professional infective agents in human material, we recommend handling calibrators and controls as potentially infective
in vitro diagnostic use only. material.
• Bovine serum albumin (BSA) used in components has been tested for BSE and found negative.
Elevated faecal calprotectin levels may be found in patients with chronic inflammatory bowel disease (IBD), and
• Avoid contact with the substrate TMB (3,3´,5,5´-Tetramethyl-benzidine).
other abdominal disorders, e.g. infectious gastro-enteritis, necrotising enterocolitis, cystic fibrosis, and colorectal
• Stop solution contains acid, classifiaction is non-hazardous. Avoid contact with skin.
carcinoma. Calprotectin in stool is a sensitive marker for IBD and is particularly useful for differentiating irritable
• Control, sample buffer, extraction medium and wash buffer contain sodium azide 0.09% as preservative. This
bowel syndrome (IBS). In addition, measurement of the calprotectin level may contribute to the evaluation of
concentration is classified as non-hazardous.
disease activity.
• Enzyme conjugate contains ProClin 300 0.05% as preservative. This concentration is classified as non-
hazardous.
SYMBOLS USED ON LABELS During handling of all reagents, controls and serum samples observe the existing regulations for laboratory safety
regulations and good laboratory practice:
V In vitro diagnostic medical device Microplate • First aid measures: In case of skin contact, immediately wash thoroughly with water and soap. Remove
Calibrator contaminated clothing and shoes and wash before reuse. If system fluid comes into contact with skin,
M Manufacturer Calibrator wash thoroughly with water. After contact with the eyes carefully rinse the opened eye with running
Calibrator
h Catalogue number Calibrator
water for at least 10 minutes. Get medical attention if necessary.
• Personal precautions, protective equipment and emergency procedures:
Calibrator Observe laboratory safety regulations. Avoid contact with skin and eyes. Do not swallow. Do not pipette by mouth.
X96 Sufficient for 96 determinations
Calibrator Do not eat, drink, smoke or apply makeup in areas where specimens or kit reagents are handled. When spilled,
g Batch code Control positive
Control negative
absorb with an inert material and put the spilled material in an appropriate waste disposal.
• Exposure controls / personal protection: Wear protective gloves of nitril rubber or natural latex.
H Use by Sample Buffer P Wear protective glasses. Used according to intended use no dangerous reactions known.
Enzyme Conjugate • Conditions to avoid: Since substrate solution is light-sensitive. Store in the dark.
8:
r
2: Temperature limitation Extraction Medium • For disposal of laboratory waste the national or regional legislation has to be observed.
TMB Substrate
w Keep away from sunlight
Stop solution
Observe the guidelines for performing quality control in medical laboratories by assaying control sera.

Wash Buffer
D Do not reuse
Ready to use
Date of manufacture
N
C CE marked according to 98/79/EC

Consult instructions for use

i
580_4 Electronic Instruction For Use: version

ORG 580_IFU_EN_QM140866_2022-03-08_4 page 1 ORG 580_IFU_EN_QM140866_2022-03-08_4 page 2

CONTENTS OF THE KIT • Do not expose reagents to heat, sun, or strong light during storage and usage.
ORG 580 X96 Sufficient for 96 determinations • Store microplate sealed and dessicated in the clip bag provided.
1 One divisible microplate consisting of 12 modules of 8 wells each. Ready to use. • Shelf life of the unopended test kit is 18 months from day of production.
Unopened reagents are stable until expiration of the kit. See labels for individual batch.
Product code on module: CP
• Diluted Wash Buffer, Sample Buffer and Extraction Medium are stable for at least 30 days when stored at 2-8°C.
1x 1.5 ml Calibrator A 15 µg/g, containing human calprotectin in a serum/buffer matrix (PBS,
We recommend consumption on the same day.
BSA, detergent, NaN3 0.09%), yellow. Ready to use.
1x 1.5 ml Calibrator B 50 µg/g; containing human calprotectin in a serum/buffer matrix (PBS,
PROCEDURAL NOTES
BSA, detergent, NaN3 0.09%), yellow. Ready to use.
• Do not use kit components beyond their expiration dates.
1x 1.5 ml Calibrator C 100 µg/g; containing human calprotectin in a serum/buffer matrix (PBS,
• Do not interchange kit components from different lots and products.
BSA, detergent, NaN3 0.09%), yellow. Ready to use.
• All materials must be at room temperature (20-28°C) prior to use.
1x 1.5 ml Calibrator D 200 µg/g; containing human calprotectin in a serum/buffer matrix (PBS,
• Extract samples and prepare all reagents. Once started, performe the test without interruption.
BSA, detergent, NaN3 0.09%), yellow. Ready to use.
• Double determinations may be done. By this means pipetting errors may become obvious.
1x 1.5 ml Calibrator E 500 µg/g; containing human calprotectin in a serum/buffer matrix (PBS,
• Perform the assay steps only in the order indicated.
BSA, detergent, NaN3 0.09%), yellow. Ready to use.
• Always use fresh sample dilutions.
1x 1.5 ml Calibrator F 1000 µg/g; containing human calprotectin in a serum/buffer matrix (PBS,
• Pipette all reagents and samples into the bottom of the wells.
BSA, detergent, NaN3 0.09%), yellow. Ready to use. • To avoid carryover or contamination, change the pipette tip between samples and different kit controls.
1x 1.5 ml Control positive, containing human calprotectin in a serum/buffer matrix (PBS, BSA, • Wash microwells thoroughly and remove the last droplets of wash buffer.
detergent, NaN3 0.09%), yellow. Ready to use. The concentration is specified on the • All incubation steps must be accurately timed.
certificate of analysis. • Do not re-use microplate wells.
1x 1.5 ml Control negative, containing human calprotectin in a serum/buffer matrix (PBS, BSA, • To avoid contamination of stool samples and assay reagents with calprotectin from the skin, suitable protective
detergent, NaN3 0.09%), yellow. Ready to use. The concentration is specified on the clothing should be worn, e.g., longer gloves, face mask.
certificate of analysis.
20 ml Sample Buffer P, containing PBS, BSA, detergent, preservative sodium azide 0.09%; PREPARATION OF REAGENTS
yellow, concentrate (5 x).
15 ml Enzyme Conjugate containing anti-Calprotectin, HRP labelled; PBS, BSA, detergent, Dilute the contents of one vial of the buffered wash solution concentrate (50x) with distilled or deionised water to a
preservative ProClin 300 0,05%, light red. Ready to use. final volume of 1000 ml prior to use.
20 ml Extraction medium; containing TBS, stabilizing protein, extraction reagent,
preservative sodium azide 0.09%; 4 x conc. Sample Buffer P: Prior to use dilute the contents (20 ml) of one vial of sample buffer 5x concentrate with distilled or
15 ml TMB Substrate; containing 3,3’, 5,5’- Tetramethylbenzidin, colorless. Ready to use. deionised water to a final volume of 100 ml.
15 ml Stop solution; contains acid. Ready to use.
20 ml Wash Buffer, containing Tris, detergent, preservative sodium azide 0.09%; 50 x conc. Extraction Medium: Prior to use dilute the contents (20 ml) of one vial of extraction medium 4x concentrate with
distilled or deionised water to a final volume of 80 ml.
MATERIALS REQUIRED
SPECIMEN COLLECTION, PREPARATION, STORAGE AND HANDLING
• Microplate reader capable of endpoint measurements at 450 nm; optional: reference filter at 620 nm
• Data reduction software Collecting stool samples
• Multi-channel dispenser or repeatable pipette for 100 μl Stool Sample Collector (SSCO) may be used.
• Vortex mixer, rocking shaker, benchtop centrifuge, optional: precision scales • Avoid contamination with toilet water containing disinfectants.
• Pipettes for 10 μl, 100 μl and 1000 μl, optional: 5 µl • Keep the collected stool sample no longer than 5 days at 2-8 °C. Alternatively, store at -20 °C.
• Laboratory timing device
• Distilled or deionised water Preparing stool sample for Extraction
• Measuring cylinder for 1000 ml and 100 ml Stool sample and have to be at room temperature.
• Plastic container for storage of the wash solution For the Alegria® Calprotectin assay the stool sample has to be diluted 1:50 in the provided.
Inhomogeneous samples should be homogenised prior to sampling, e.g. by using an inoculation loop.
AUXILIARY REAGENTS For liquid stool samples we recommend to weigh out sample.
• ORG 282 Stool Extraction Tubes; containing 100 tubes.
• SSCO Stool Sample Collector Extraction of Calprotectin using Stool Extraction Tube (ORG 282)
• F5179 Extraction medium, 20 ml, 4 x conc. Use Stool extraction Tubes according to Instruction for Use ORG 282
These auxiliaries are available separately. • Fill 750 µl into the transparent tube to gain a 1:50 dilution.
• Extraction
AUTOMATION Vortex 30 sec at 1800 rpm to remove stool sample from the notches of the spatula.
This ELISA assay is suitable for use on open automated ELISA processors. Each assay has to be validated on the Further homogenize for 15 min at top speed on a rocking shaker.
respective automated system. Detailed information is provided upon request. Open tube. Transfer the homogenate to a microtube. Centrifuge for 2 min at 3000xg.
Transfer clear fluid to another microtube and test for Calprotectin immediately.

STORAGE AND STABILITY Extraction of Calprotectin using other tubes

• Store test kit at 2-8°C in the dark. • Weigh out empty tube.

ORG 580_IFU_EN_QM140866_2022-03-08_4 page 3 ORG 580_IFU_EN_QM140866_2022-03-08_4 page 4

 Pick stool sample and transfer to tube. Weigh out and dExtraction of Calprotectin using other tubesetermine net Test results are valid if the optical densities at 450 nm for calibrators / controls and the results for controls comply
weight of sample. with the reference ranges indicated on the Certificate of Analysis enclosed in each test kit.
• Fill into the tube: 49-times net weight of sample needed to gain a 1:50 dilution If these quality control criteria are not met the assay run is invalid and should be repeated.
stool sample + stool sample + CALCULATION OF RESULTS
15 mg + 0.75 ml 60 mg + 2.9 ml For quantitative results plot the optical density of each calibrator versus the calibrator concentration to create a
20 mg + 1.0 ml 70 mg + 3.4 ml calibration curve. The concentration of patient samples may then be estimated from the calibration curve by
30 mg + 1.5 ml 80 mg + 3.9 ml interpolation.
40 mg + 2.0 ml 90 mg + 4.4 ml Using data reduction software a 4-Parameter-Fit with lin-log coordinates for optical density and concentration is the
50 mg + 2.5 ml 100 mg + 4.9 ml data reduction method of choice.
Close tube. Vortex 30 sec at 1800 rpm. Further homogenize for 15 min at top speed on a rocking shaker.
Open tube. Transfer the homogenate to a microtube. Centrifuge for 2 min at 3000xg. Extended measuring range*
Transfer clear fluid to another microtube and test for Calprotectin immediately. It is possible to extend the measuring range from 15-1000 µg/g to 45-3000 µg/g by following this procedure:
• dilute extrated stool sample 1:300, use 100 µl per well
Such an extracted stool sample may be stored at 2-8 °C for up to 5 days or at -20 °C for up to 4 • use calibrators / controls as usual and perform the assay as usual,
months. The extracted stool sample is not stable at room temperature! • for calculation of results assign the calibrators the following concentrations:
A: 45 µg/g, B: 150 µg/g, C: 300 µg/g, D: 600 µg/g, E: 1500 µg/g, F: 3000 µg/g

Dilution of extracted stool samples

Dilute extracted stool samples 1:100 before the assay: PERFORMANCE CHARACTERISTICS
Put 990 µl of prediluted sample buffer in a polystyrene tube and add 10 µl of extracted stool sample. Mix well.
Note: Calibrators / Controls are ready to use and need not be diluted. Calibration
The assay system is calibrated using a Calprotectin reference preparation purified from human neutrophils.
For extended measuring range*
If the Calprotectin value is > 1000 µg/g, we recommend testing the extracted stool sample again in higher dilution: Measuring range
Put 1495 µl of prediluted sample buffer in a polystyrene tube and add 5 µl of extracted stool sample to obtain a The calculation range of this ELISA assay is 15 - 1000 µg/g
dilution of 1:300. Mix well.
Note: Calibrators / Controls are ready to use and need not be diluted. Expected values
In a normal range study with samples from healthy donors the following ranges have been established with this
TEST PROCEDURE ELISA assay: Cut-off 50 µg/g
Prepare enough microplate modules for all calibrators / controls and patient samples.
1. Pipette 100 µl of calibrators, controls and prediluted extracted stool samples into the wells.
Interpretation of results
Incubate for 30 minutes at room temperature (20-28 °C). Normal range < 50 µg/g
Discard the contents of the microwells and wash 3 times with 300 µl of wash solution. Slightly elevated values 50 - 200 µg/g
Significantly elevated values > 200 µg/g
2. Dispense 100 μl of enzyme conjugate into each well.
Incubate for 15 minutes at room temperature.
Discard the contents of the microwells and wash 3 times with 300 μl of wash solution. Calprotectin level in the normal range: gastrointestinal inflammation nearly ruled out; further invasive diagnostic
measures are generally not required.
3. Dispense 100 μl of TMB substrate solution into each well. Calprotectin level slightly elevated: low level of inflammatory activity in the gastrointestinal tract (e.g. IBD in
Incubate for 15 minutes at room temperature remission); repeated test and further diagnostic measures are recommended.
4. Add 100 μl of stop solution to each well of the modules Calprotectin level significantly elevated: active organic disease of the gastrointestinal tract; further intensive
Incubate for 5 minutes at room temperature. diagnostic tests and treatment by a specialised gastroenterologist are urgently advised.
Read the optical density at 450 nm (reference 600-690nm) and calculate the results.
The developed colour is stable for at least 30 minutes. Read during this time. Linearity
Example for a pipetting scheme: Patient samples containing high levels of Calprotectin were serially diluted in sample buffer to demonstrate the
1 2 3 4 5 6 7 8 9 10 11 12 dynamic range of the assay and the upper / lower end of linearity. Activity for each dilution was calculated from the
A A P1 calibration curve using a 4-Parameter-Fit with lin-log coordinates. *Extended measuring range
.
B B P2
Sample Dilution Observed Expected O/E
C C P3
.. .. µg/g µg/g [%]
D D
1 1:100 896.9 896.9 100
E E
. 1:200 526.7 448.5 117
F F
. 1:400 246.0 224.2 110
G C+
. 1:800 118.1 112.1 105
H C-
. 1:1600 55.8 56.1 100
2 1:100 485.3 485.3 100
P1, ... patient sample A-F calibrators C+, C- controls
. 1:200 247.1 242.7 102
VALIDATION . 1:400 121.5 121.3 100
. 1:800 60.4 60.7 100

ORG 580_IFU_EN_QM140866_2022-03-08_4 page 5 ORG 580_IFU_EN_QM140866_2022-03-08_4 page 6

 . 1:1600 27.1 30.3 89 drugs. NSAID can lead to elevated concentrations of fecal calprotectin. In these cases reevaluation of fecal
3* 1:300 2354.0 2354.0 100 calprotectin after stopping NSAIDs should be considered.
. 1:600 1083.9 1177.0 92 Calprotectin is present throughout the entire body and is highly expressed during differentiation of the epidermis in
. 1:1200 547.1 588.5 93 particular. When handling kits and samples, suitable protective measures should be taken to avoid contamination
. 1:2400 261.3 294.3 89 by calprotectin from skin surfaces.

REFERENCES
Limit of detection 1. Burri; Beglinger: “Faecal calprotectin – a useful tool in the management of inflammatory bowel disease”. Swiss
Lowest detectable concentration: 17.5 µg/g Med Wkly. 2012; 142: w13557
2. Van Rheenen et al.: “Faecal calprotectin for screening of patients with suspected inflammatory bowel disease:
Reproducibility diagnostic meta-analysis”. BMJ. 2010; 341: c3369
Intra-assay precision: Coefficient of variation (CV) was calculated for each of three samples from the results of 24 3. Johne; Fagerhol et al.: “Functional and clinical aspects of the myelomonocyte protein calprotectin”. J Clin
determinations in a single run. Results for precision-within-assay are shown in the table below. Pathol: Mol Pathol. 1997; 50: 113-123
Inter-assay precision: Coefficient of variation (CV) was calculated for each of three samples from the results of 6 4. Berni et al.: “Faecal calprotectin is a useful diagnostic tool in pediatric gastroenterology”. Ital J Pediatr. 2005;
determinations in 5 different runs. Results for run-to-run precision are shown in the table below. 31: 89-94
*Extended measuring range 5. Canani et al.: "Diagnostic value of faecal calprotectin in paediatric gastroenterology clinical practice“. Digestive
. and Liver Disease. 2004; 36: 467-470
Intra-Assay Inter-Assay 6. Costa et al.: “Role of faecal calprotectin as non-invasive marker of intestinal inflammation”. Digestive and Liver
Sample Mean . Sample Mean . Disease. 2003; 35: 642-647
. µg/g % CV . µg/g % CV 7. Chapaiboon et al.: "Calprotectin S100A9 calcium binding loops I and II are essential for keratinocyte resistance
1 52.6 3.7 1 81.3 11.6 to bacterial invasion“. The Journal of biological Chemistry. 2009; Vol 284; No 11: 7078-7090
2 437.7 3.9 2 157.1 13.5 8. Costa et al.: “Calprotectin is a stronger predictive marker of relapse in ulcerative colitis than in Crohn´s
3 845.3 5.8 3 796.6
. 12.0
. disease”. Gut. 2005; 54: 364-368
4* 1340.0 4.8 4* 1358.3 4.6 9. Fagerberg et al.: "Fecal calprotectin levels in healthy children studied with an improved assay“. Journal of
Pediatric Gastroenterology and Nutrition. 2003; 37: 468-472
10. Montalto et al.: “Fecal calprotectin in first-degree relatives of patients with ulcerative colitis“. Am J
Interfering substances Gastroenterol. 2006; 101: 1-5
The following substances were tested per 15 mg of stool sample and were found to be non-interfering: Vancomycin 11. Roseth et al.: "Normalization of faecal calprotectin: a predictor of mucosal healing in patients with inflammatory
hydrochlorid 0.201 mg, Ciprofloxacin 0.15 mg, Prevazid 0.006 mg, Azathioprine 0.021 mg, Prednisone 0.003 mg, bowel disease“. Scand J Gastroenterol. 2004; 39: 1017-1020
Pentasa 0.399 mg, Vitamin A 2.4 UI, Vitamin C 0.015 mg, Vitamin E 0.03 mg, Vitamin D3 0.33 UI, 12. Whitehead et al.: "Between-assay variability of faecal calprotectin enzyme-linked immunosorbent assay kits”.
Hemoglobin 1.749 mg. Annals of Clinical Biochemistry. 2013; 50: 53-61
The following microorganisms were tested at 1.1 x 107 CFU per 15 mg of stool sample and were found to be non- 13. NICE Diagnostics guidance 11, Oct. 2013, Faecal calprotectin diagnostic tests for inflammatory diseases of the
interfering: E. coli, Klebsiella pneumoniae, Salmonella enterica, Shigella flexneri, Yersinia enteroclitica . bowel.
14. Layer et al.: "S3-Leitlinie Reizdarmsyndrom: Definition, Pathophysiologie, Diagnostik und Therapie.
Study results Gemeinsame Leitlinie der Deutschen Gesellschaft für Verdauungs- und Stoffwechselkrankheiten (DGVS) und
Study population n n pos % der Deutschen Gesellschaft für Neurogastroenterologie und Motilität (DGNM)". Z. Gastroenterol. 2011; 49:237
Inflammatory bowel disease 185 181 97.8 -293
Abdominal disorders 142 10 7.0 15. Carroccio et al.:"Diagnostic accuracy of fecal calprotectin assay in distinguishing organic causes of diarrhea
Healthy donors 18 0 0.0 from irritable bowel syndrome: a prospective study in adults and children." Clin. Chem. 2003; 49:6: 861-867.
16. Montalto et al.: “Role of fecal calprotectin in gastrointestinal disorder“. Eur Rev Med Pharmacol Sci. 2013; 17:
1569-1582
Diagnostic result 17. Tibble et al.: “High prevalence of NSAID enteropathy as shown by simple faecal test”. Gut. 1999; 45: 362-366.
POS NEG 18. Oyaert et al.: "Analytical performance and diagnostic accuracy of six different faecal calprotectin assays in
ORG 580 POS 181 10 inflammatory bowel disease". Clin Chem Lab Med. 2017; 55: 1564-1573
NEG 4 150
. 185 160 345 .
Sensitivity: 97.8 %
Notice to the user (European Union):
Specificity: 93.8 %
Any serious incident that has occurred in relation to the device shall be reported to the manufacturer and the
Overall agreement: 95.9 %
competent authority of the EU Member State in which the user and/or the patient is established .

LIMITATIONS OF THE PROCEDURE Change Control

This assay is a diagnostic aid. A definite clinical diagnosis should not be based on the results of a single test, but Former version: ORG 580_IFU_EN_QM140866_2018-01-02_3 Reason for revision: Note to prevent cross contamination added
should be made by the physician after all clinical and laboratory findings have been evaluated concerning the entire
clinical picture of the patient. Also every decision for therapy should be taken individually.
The above pathological and normal reference ranges in patient stool samples should be regarded as
recommendations only. Each laboratory should establishe its own ranges according to ISO 15189 or other
applicable laboratory guidelines.
Non-steroidal anti-inflammatory drugs (NSAIDs) induce enteropathy in up to 20-65 % of patients receiving these
ORG 580_IFU_EN_QM140866_2022-03-08_4 page 7 ORG 580_IFU_EN_QM140866_2022-03-08_4 page 8
ORG 580_IFU_EN_QM140866_2022-03-08_4 page 9