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Calprotectin (S100A8/A9) ELISA for Human Stool (ImmuChrom)

IC7300
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Intended Use

The Calprotectin (S100A8/A9) ELISA Kit is an immunoassay intended for the quantitative determination of calprotectin in human stool. For research use only.

Description

Human calprotectin is a dimer which consists of the subunits S100A8 (10.835 kDa) and S100A9 (13.242 kDa). The monomers are able to bind calcium. The complex is located in the cytosol of neutrophils and is excreted to the intestine while inflammation (1). The concentration of fecal samples correlates with the severity of inflammatory processes in the intestine. The complex is resistant against enzymatic degradation. The measurement of fecal calprotectin represents an easy non-invasive analysis of intestinal inflammation with the possibility to differentiate between noninflammatory irritable bowel disease and intestinal inflammation accompanied with morphological alteration of the intestinal mucosa (2,3).

In case of viral or bacterial infections of the gut the concentration of calprotectin in feces is increased (4). For this reason, to determine if increase is do to chronic inflammatory disease, always first excluded possible infectious cause (e.g. detection of pathogens, detection of pathogen-specific antibodies).

The use of non-steroidal anti-inflammatory drugs (e.g. aspirin, ibuprofen, diclofenac) or COX-2 inhibitors (e.g. celecoxib) can lead to enteropathies that lead to an increase in the calprotectin level in the stool (5,6). Before carrying out the determination, the corresponding medication should therefore not be taken for a period of 14 days if possible so as not to influence the measurement of the degree of intestinal inflammation (7).

The ImmuChrom complete calprotectin kit allows an easy, rapid and precise quantitative determination of calprotectin in biological samples. The kit includes all reagents ready to use for preparation of the samples.

Regulatory Status

Research Use Only (RUO)

Type

Sandwich ELISA, HRP-labelled antibody

Other names

fecal calprotectin, faecal calprotectin, S100A8/A9, S100A8/S100A9, MRP8-MRP14, cystic fibrosis antigen, L1, 60BB antigen, 27E10 antigen.

Principle of Method

The calprotectin ELISA test determines human calprotectin according to the “sandwich”-principle. Calprotectin in sample, standard and controls binds to antibodies, which are coated to the microtiterplate. After a washing step a peroxidase labeled detection antibody is added. A second washing step is followed by the addition of the substrate which is converted to a colored product by the peroxidase. The reaction is terminated by the addition of an acidic stop solution. The optical densities are read at 450 nm (against the reference wavelength 620 nm) in a microtiterplate reader. The calprotectin concentration can be calculated from the standard curve.

Calibration: The test system is calibrated from a reference preparation of recombinant and purified calprotectin from E. coli.

Sample Types

Stool

Sample Volume

Extraction in stick vials

In a stool sample extraction vial mix 15 mg stool with 1.5 ml extraction buffer (EXT), then vortex it until the mixture is homogenous. Transfer the resulting slurry to a plastic vial and centrifuge it for 10 min at 3000 xg. The supernatant is diluted 1:38.5 in sample buffer (SAMPLEBUF). We recommend 20 µl supernatant to mix with 750 µl sample buffer. 100 µl of the dilution are used in the test per well.

Please use only plastic vials and no glass vials

Standard Curve

Example of a standard curve with the Immuchrom Calprotectin Human ELISA kit for stool samples

Calibration Range:  3 - 208 ng/ml

Limit of Detection

0.997 ng/ml

For the determination the zero-standard was measured 20 times. The 3-fold standard deviation was added to the mean value of the optical density. The respective concentration was read from the standard curve.

Intra-assay (Within-Run) C.V.

<10 %

Inter-assay (Run-to-Run) C.V.

< 15 %

Resources

 Ilex Life Sciences LLC is an official distributor of Immuchrom GmbH.

References to Summary

• Roseth AG, et al. 1999. Correlation between faecal excretion of indium-111- labelled granulocytes and calprotectin, a granulocyte marker protein, in patients with inflammatory bowel disease. Scand J Gastroenterol. 34(1): 50-5

• Sidler MA, et al. 2008. Fecal S100A12 and fecal calprotectin as noninvasive markers for inflammatory bowel disease. Inflamm Bowel Dis. 14(3): 359-66

• Sipponen T, Kolho KL 2015. Fecal calprotectin in diagnosis and clinical assessment of inflammatory bowel disease. Scand J Gastroenterol. 50(1): 74- 80

• Chen CC, et al. 2012. Fecal calprotectin as a correlative marker in clinical severity of infectious diarrhea and usefulness in evaluating bacterial or viral pathogens in children. J Pediatr Gastroenterol Nutr. 55(5): 541-7

• Tibble JA, et al. 1999. High prevalence of NSAID enteropathy as shown by a simple faecal test. Gut. 45(3): 362-6

• Goldstein JL, et al. 2007. Small bowel mucosal injury is reduced in healthy subjects treated with celecoxib compared with ibuprofen plus omeprazole, as assessed by video capsule endoscopy. Aliment Pharmacol Ther. 25(10): 1211-22

• Rendek Z, et al. 2016. Effect of oral diclofenac intake on faecal calprotectin. Scand J Gastroenterol. 51(1): 28-32

• Bressler B, et al. 2015. Clinicians´ guide to use of fecal calprotectin to identify and monitor disease activity in inflammatory bowel disease. Can J Gastroenterol Hepatol. 29(7): 369-72

 Ilex Life Sciences LLC is an official distributor of Immuchrom GmbH products.