PROCR ELISA Kit

Catalog No. ABIN456435

Product Details PROCR ELISA Kit

Target: PROCR (Protein C Receptor, Endothelial (PROCR))

Binding Specificity: Soluble

Reactivity: Mouse

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.312-20 ng/mL

Minimum Detection Limit: 0.312 ng/mL

Application: ELISA

Purpose: This immunoassay kit allows for the specific measurement of mouse soluble endothelial protein C receptor, sEPCR concentrations in cell culture supernates, serum and plasma.

Sample Type: Cell Culture Supernatant, Serum, Plasma

Analytical Method: Quantitative

Specificity: This assay recognizes recombinant and natural mouse sEPCR.

Cross-Reactivity (Details): No significant cross-reactivity or interference was observed.

Sensitivity: The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.

Characteristics: Mus musculus,Mouse,Endothelial protein C receptor,Activated protein C receptor,APC receptor,Centrocyclin,Centrosomal protein CCD41,Endothelial cell protein C receptor,Procr,Epcr,CD201

Components: Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1x120µl), Detection Reagent B (1x120µl), Wash Buffer(25 x concentrate) (1x30ml), Substrate (1x10ml), Stop Solution (1x10ml)

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: This assay employs the quantitative sandwich enzyme immunoassay technique. A antibody specific for sEPCR has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any sEPCR present is bound by the immobilized antibody. An enzyme-linked antibody specific for sEPCR 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 sEPCR 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 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 10,000 pg/mL. Allow the standard to sit for a minimum of 15 minutes 3 with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard 10,000 pg/mL. The Sample Diluent serves as the zero standard (0 pg/mL). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.

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.

Assay Procedure:

Allow all reagents to reach room temperature. Arrange and label required number of strips.
1. Prepare all reagents, working standards and samples as directed in the previous sections.
2. Add 100 uL of Standard, Control, or sample per well. Cover with the adhesive strip. Incubate for 2 hours at 37 °C.
3. Remove the liquid of each well, don’t wash.
4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 °C.
7. Repeat the aspiration/wash as in step
5. 8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Important Note:
1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
3. Duplication of all standards and specimens, although not required, is recommended.
4. When mixing or reconstituting protein solutions, always avoid foaming.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

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 y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the sEPCR concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. 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

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 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 Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.

Target Details for PROCR

Target: PROCR (Protein C Receptor, Endothelial (PROCR))

Alternative Name: Procr (PROCR Products)

Synonyms: PROCR ELISA Kit, CCCA ELISA Kit, CCD41 ELISA Kit, EPCR ELISA Kit, AI325044 ELISA Kit, Ccca ELISA Kit, Ccd41 ELISA Kit, Epcr ELISA Kit, protein C receptor ELISA Kit, protein C receptor, endothelial ELISA Kit, PROCR ELISA Kit, Procr ELISA Kit

Background: The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. The Endothelial Protein C Receptor (EPCR, also referred to as CCD41 or CD201) is a 25 kD Type 1 transmembrane protein expressed on endothelial cells. EPCR is a ligand for Protein C and plays an important role in augmenting Protein C activation by the thrombin-thrombomodulin complex and in regulating blood coagulation and inflammation. A soluble form of EPCR (sEPCR) has recently been detected in normal mouse plasma and has been shown to bind protein C and APC with an affinity similar to that of intact membrane-bound EPCR. In healthy individuals, sEPCR circulates at a concentration of 2.5 nM, a level that can increase up to 5-fold in patients with sepsis or systemic lupus erythematosus. In contrast to membrane-bound EPCR, sEPCR inhibits protein C activation over large vessel endothelium in culture. This presumably reflects competition between the sEPCR and cell surface EPCR. sEPCR also inhibits APC anticoagulant activity, but the mechanism responsible for this inhibition remains unclear. Since EPCR interacts with the membrane-binding Gla domain of protein C, it is possible that binding to sEPCR and phospholipid is mutually exclusive. Alternatively, sEPCR could mask the factor Va-binding site on APC or alter the macromolecular substrate specificity of the enzyme by altering the conformation of the extended substrate binding pocket.