Purpose | This immunoassay kit allows for the specific measurement of rat Intercellular Adhesion Molecule (ICAM-1) concentrations in cell culture supernates, serum, tissue homogenates and plasma. |
Sample Type | Cell Culture Supernatant, Serum, Tissue Homogenate, Plasma |
Analytical Method | Quantitative |
Detection Method | Colorimetric |
Specificity | This assay recognizes recombinant and natural rat ICAM-1. |
Cross-Reactivity (Details) | No significant cross-reactivity or interference was observed. |
Sensitivity | 21pg/ml |
Characteristics | Rattus norvegicus,Rat,Intercellular adhesion molecule 1,ICAM-1,Icam1,Icam-1,CD54 |
Components |
Reagent (Quantity):
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Material not included | Microplate reader. Pipettes and pipette tips. EP tube Deionized or distilled water. |
Alternative Name | Icam1 (ICAM1 ELISA Kit Abstract) |
Background | Adhesion molecules mediate the interaction of cells with the extracellular matrix and with other cells. The immunoglobulin superfamily of proteins contains a large class of adhesion molecules with multiple immunoglobulin-like domains. ICAM is a member of this family. It is a 90 kDa type-I transmembrane glycoprotein with five Ig-like extracellular domains. The most important ligands for ICAM-1 are the _2 integrins LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), which are expressed on leukocytes. ICAM-1 thus mediates the adhesion of leukocytes to ICAM-1-expressing cells. ICAM-1 also binds fibrinogen, hyaluronan, Rhinoviruses, Plasmodium falciparum-infected erythrocytes and CD43 (sialophorin) ICAM-1 is either a transmembrane protein (mICAM-1) or soluble (sICAM-1). mICAM-1 is expressed on endothelial and epithelial cells, lymphocytes, monocytes, eosinophils, keratinocytes, dendritic cells, hematopoietic stem cells, hepatocytes and fibroblasts. Regulation of ICAM-1 expression is cell specific. Up-regulation generally is by inflammatory cytokines (TNF-alpha, IFN-gammaand IL-1) and down-regulation generally is by anti-inflammatory agents (e.g.glucocorticoids). One important, well-characterized function of ICAM-1 is immune-cell trafficking. At sites of inflammation, inflammatory cytokines induce up-regulation of ICAM-1 expression on vascular endothelial cells and activation of leukocyte integrins (LFA-1 and Mac-1). This leads to adhesion of leukocytes to the local endothelium, an essential step in migration of leukocytes to the site of inflammation. ICAM-1 has been reported in serum, cerebrospinal fluid and bronchoalveolar lavage. ICAM-1 likely arises by proteolytic cleavage of mICAM-1, synthesis from an alternatively spliced message has not been found. In general, elevated levels of serum ICAM-1 appear to be associated with inflammatory conditions and certain malignancies. It has, however, been pointed out that in inflammatory conditions, where the ligands LFA-1 and Mac-1 are likely to be activated, binding and clearance of ICAM-1 might be enhanced, so that a reciprocal relationship between ICAM-1 levels and inflammation also is possible. |
Gene ID | 2902 |
Pathways | Cellular Response to Molecule of Bacterial Origin, Regulation of Actin Filament Polymerization, Carbohydrate Homeostasis, Regulation of Leukocyte Mediated Immunity, Thromboxane A2 Receptor Signaling |
Sample Volume | 100 μL |
Plate | Pre-coated |
Protocol | This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for ICAM-1 has been pre-coated onto a microplate. Standards and samples are 2 pipetted into the wells and any ICAM present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for ICAM-1 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of ICAM-1 bound in the initial step. The color development is stopped and the intensity of the color is measured. |
Reagent Preparation |
Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard. The Sample Diluent serves as the zero standard (0 ng/ml). |
Sample Collection | Cell culture supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay. Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, heart and lung tissue from eight mice or one rat was rinsed with 1X PBS to remove excess blood, homogenized in 20 mL of 1X PBS and stored overnight at ≤ -20 °C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 x g. Remove the supernate and assay immediately or aliquot and store at ≤ -20 °C. |
Assay Procedure |
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37 °C directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4 °C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments. |
Calculation of Results |
Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the SAA concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 13.0. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. |
Restrictions | For Research Use only |
Handling Advice |
1. The kit should not be used beyond the expiration date on the kit label. 2. Do not mix or substitute reagents with those from other lots or sources. 3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause 3 variation in binding. 4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded. |
Storage | 4 °C/-20 °C |
Storage Comment | The Assay Plate, Standard, Detection Reagent A and Detection Reagent B should be stored at -20°C upon being received. After receiving the kit , Substrate should be always stored at 4°C. |
Product cited in: |
Ragab, Abdallah, El-Abhar: "Cilostazol renoprotective effect: modulation of PPAR-?, NGAL, KIM-1 and IL-18 underlies its novel effect in a model of ischemia-reperfusion." in: PLoS ONE, Vol. 9, Issue 5, pp. e95313, 2014 (PubMed).
El-Naga: "Pre-treatment with cardamonin protects against cisplatin-induced nephrotoxicity in rats: impact on NOX-1, inflammation and apoptosis." in: Toxicology and applied pharmacology, Vol. 274, Issue 1, pp. 87-95, 2013 (PubMed). |