The human Calprotectin ELISA kit is to be used for the in vitro quantitative determination of human Calprotectin in feces, plasma, serum, sputum and urine samples. This kit is intended for laboratory research use only and is not for use in diagnostic or therapeutic procedures. The kit cannot be used for quantification of the individual MRP-8 (S100A8) or MRP-14 (S100A9) proteins. The analysis should be performed by trained laboratory professionals.
Contents of Kit
1. Wash buffer 40×: 1 vial (30 ml), Colorless 2. Dilution buffer A 10×: 1 vial (30 ml), Green 3. Dilution buffer B 10×: 2 vials (15 ml), Colorless 4. Standard: 2 vials, lyophilized, White 5. Tracer, biotinylated: 1 vial, 1 ml lyophilized, White 6. Streptavidinperoxidase 100×: 1 tube, 0.25 ml in solution, Brown 7. TMB substrate: 1 vial (11 ml), Brown 8. Stop solution: 1 vial (22 ml), Red 9. 12 Microtiter strips, pre-coated: 1 plate Note:Upon receipt, store individual components at 2 – 8 °C. Do not freeze. Do not use components beyond the expiration date printed on the kit label. The standard and tracer in lyophilized form and the streptavidin-peroxidase in concentrated solution are stable until the expiration date indicated on the kit label, if stored at 2 – 8 °C. The exact amount of the standard is indicated on the label of the vial and the Certificate of Analysis. The standard is single use. After reconstitution the standard cannot be stored. Once reconstituted the tracer is stable for 1 month if stored at 2 – 8 °C. The streptavidin-peroxidase can only be stored in concentrated solution and is not stable when stored diluted. Upon receipt, foil pouch around the plate should be vacuum-sealed and unpunctured. Any irregularities to aforementioned conditions may influence plate performance in the assay. Return unused strips immediately to the foil pouch containing the desiccant pack and reseal along the entire edge of the zip-seal. Quality guaranteed for 1 month if stored at 2 – 8 °C.
Storage
Store the complete kit at 2 – 8 °C. Under these conditions, all components are stable until the expiration date (see label on the box).
Precision
The intra-assay precision and reproducibility was tested with four samples using three different dilutions. Samples were tested twice in quadruplicate by the same technician on the same day. The inter-assay precision and reproducibility was tested with four samples in duplicate, using two different batches that were performed by two technicians on two separate days.
Detection Range
1.6 - 100 ng/ml
Detection Limit
1.6 ng/ml
General Description
Calprotectin, also known as MRP-8/MRP-14 or S100A8/A9 heterocomplex, is formed out of the calcium-binding, migration inhibitory factor-related proteins, MRP-8 (S100A8) and MRP-14 (S100A9). The expression of these proteins is largely confined to the cytosol of neutrophils and monocytes. The complex formation of these proteins is calcium-dependent. Calprotectin comprises 60% of the cytoplasmic protein fraction of circulating polymorphonuclear granulocytes and is also found in monocytes, macrophages and ileal tissue eosinophils. Peripheral blood monocytes carry the antigen extra- and intracellularly, neutrophils only intracellularly. Calprotectin has antibacterial, antifungal, immunomodulating and antiproliferative effects. Furthermore, it is a potent chemotactic factor for neutrophils. Plasma concentrations are elevated in diseases associated with increased neutrophil activity. During intestinal wall inflammation, granulocytes transmigrate through the intestinal wall. Therefore Calprotectin is also detectable in feces. Several investigations report that fecal Calprotectin is significantly increased in intestinal diseases such as inflammatory bowel disease (IBD), Crohn´s disease, ulcerative colitis and colon cancer. In the literature it is described that normal human plasma contains a Calprotectin concentration ranging from ~100 to 3,000 ng/ml.
Standard Curve
Citations
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IL-1 Mediates Tissue-Specific Inflammation and Severe Respiratory Failure in COVID-19
Renieris G, Karakike E, Gkavogianni T, Droggiti DE, Stylianakis E, Andriopoulou T, Spanou VM, Kafousopoulos D, Netea MG, Eugen-Olsen J, Simard J, Giamarellos-Bourboulis EJ
Applications: ELISA Reactive species: Human
"Abstract: Acute respiratory distress syndrome (ARDS) in COVID-19 has been associated with catastrophic inflammation. We present measurements in humans and a new animal model implicating a role in danger-associated molecular patterns. Calprotectin (S100A8/A9) and high-mobility group box 1 (HMGB1) were measured in patients without/with ARDS, and admission calprotectin was associated with soluble urokinase plasminogen activator receptor (suPAR). An animal model was developed by intravenous injection of plasma from healthy or patients with COVID-19 ARDS into C57/BL6 mice once daily for 3 consecutive days. Mice were treated with one anti-S100A8/A9 antibody, the IL-1 receptor antagonist anakinra or vehicle, and Flo1-2a anti-murine anti-IL-1α monoclonal antibody or the specific antihuman IL-1α antibody XB2001 or isotype controls. Cytokines and myeloperoxidase (MPO) were measured in tissues. Calprotectin, but not HMGB1, was elevated in ARDS. Higher suPAR indicated higher calprotectin. Animal challenge with COVID-19 plasma led to inflammatory reactions in murine lung and intestines as evidenced by increased levels of TNFα, IL-6, IFNγ, and MPO. Lung inflammation was attenuated with anti-S100A8/A9 pre-treatment. Anakinra treatment restored these levels. Similar decrease was found in mice treated with Flo1-2a but not with XB2001. Circulating alarmins, specifically calprotectin, of critically ill COVID-19 patients induces tissue-specific inflammatory responses through an IL-1-mediated mechanism. This could be attenuated through inhibition of IL-1 receptor or of IL-1α." Article snippet: Calprotectin (S100A8/A9), high-mobility group box 1 (HMGB1), suPAR, and ferritin were measured by enzyme immunosorbent assays (*, Jena, Germany and Creative Diagnostics, Shirley, NY, USA; suPARnostic, *, Lyngby, Denmark, and *, Mainz, Germany, respectively)
Figure 1. Concentrations of calprotectin (S100A8/A9) were measured in the plasma from HVs.
Figure 2. Concentrations of calprotectin in 20 patients with suPAR less than 6 ng/mL and in 20 patients with suPAR 6 ng/mL or more.
Background
Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is characterized by chronic remitting inflammatory flare-ups within and outside the gastrointestinal tract. IBD is caused by an inappropriate immune response to intestinal commensals in genetically susceptible individuals, resulting in inflammation and intestinal ulceration. Calprotectin (CP) is a clinical biomarker of inflammatory bowel disease with immunomodulatory functions. CP belongs to the evolutionarily conserved family of calcium-binding S100 leukocyte proteins, which in mammals consists of two monomers (S100A8 and S100A9). As early as the last century, researchers identified a protein complex under different inflammatory conditions, which subsequently became known as calprotectin, with the main emphasis on the protein's Ca2+-binding properties and its antifungal activity against Candida albicans.
CP is an abundant cell membrane protein complex (composed of S100A8 and S100A9) that is constitutively expressed in neutrophils, accounting for approximately 45% of total cytoplasmic proteins. In addition, CP is also constitutively expressed in monocytes, dendritic cells, activated macrophages, oral keratinocytes, and squamous mucosal epithelium, and its expression can be specifically induced during inflammation. Both subunits, S100A8 and S100A9, have a wide range of intracellular and extracellular immunoregulatory properties, and the S100A8/S100A9 complex controls the intracellular pathways of innate immune cells and orchestrates inflammatory responses. CP modulates cytoskeletal rearrangement, recruits leukocytes, and promotes arachidonic acid transport to sites of inflammation. In addition, nuclear S100A9/CP acts as a coactivator to modify transcription during inflammatory processes and malignant transformation. Extracellular Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE) mediate the extracellular function of CP. However, extracellular CP can form complex protein conformations with different biological functions and equivalent receptors that may not be explained by these signaling pathways.
Figure 1. Calprotectin in the control of gut inflammation (Source: Jukic A, et al. 2021)
The efficacy of fecal CP as a marker has been investigated in various areas of IBD diagnosis and treatment, including the differentiation of IBD from irritable bowel syndrome (IBS), the assessment of endoscopic activity of the disease, the assessment of histologic activity of the disease, and the prediction of disease relapse and response to therapy. Fecal CP has relatively high sensitivity and specificity in differentiating IBD from IBS, but lower sensitivity and specificity in predicting endoscopic and histologic remission. Therefore, despite its ease of measurement, fecal CP cannot be considered a reliable alternative to colonoscopy for assessing endoscopic activity in IBD.
Alternative Names
Human CP ELISA
References
1. Khaki-Khatibi F, et al. Calprotectin in inflammatory bowel disease. Clin Chim Acta. 2020 Nov;510:556-565.
2. Jukic A, et al. Calprotectin: from biomarker to biological function. Gut. 2021 Oct;70(10):1978-1988.
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Figure 1. Concentrations of calprotectin (S100A8/A9) were measured in the plasma from HVs. 
Figure 2. Concentrations of calprotectin in 20 patients with suPAR less than 6 ng/mL and in 20 patients with suPAR 6 ng/mL or more. 
Figure 1. Calprotectin in the control of gut inflammation
