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C-Reactive Protein (CRP) ELISA Kit

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Rat (Rattus)
Methode Type Competition ELISA
Minimum Detection Limit 70 ng/mL
Pubmed 6 references available
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Quantity 96 tests
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Purpose The AssayMax Rat CRP ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of rat CRP in plasma, serum and cell culture supernatants
Sample Type Plasma, Cell Culture Supernatant
Detection Method Colorimetric
Components Rat CRP Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against rat CRP. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. 1 Rat CRP Standard: Rat CRP in a buffered protein base (25 g, lyophilized). Biotinylated Rat CRP: 1 vial, lyophilized. EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (90 l). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).
Material not included Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel pipette) Deionized or distilled reagent grade water
Background C-Reactive Protein (CRP) is a liver protein composed of five identical nonglycosylated subunits, with a total molecular weight of 105 kDa. CRP has a variety of powerful effects related to immunology, inflammation, and coagulation. As a marker of low-level inflammation, CRP appears to predict future cardiovascular disease events among apparently healthy individuals. High plasma concentration of CRP was associated with increased risk of stroke, myocardial infarction, and peripheral vascular disease. CRP has also been associated with increased risks of fatal coronary events among high-risk male smokers and incident coronary disease among the elderly. Studies have established the prognostic usefulness of CRP in the setting of angina. Originally used as a marker of acute inflammation, CRP has become a leading candidate as the measure of choice for estimating the inflammatory component of cardiovascular disease risk.
Research Area Serum/Plasma Proteins, Inflammation
Sample Volume 25 μL
Assay Time < 3 h
Plate Pre-coated,Strips (12 x 8)
Protocol This assay employs a quantitative competitive enzyme immunoassay technique that measures rat CRP in less than 3 hours. A polyclonal antibody specific for rat CRP has been pre-coated onto a 96-well microplate with removable strips. CRP in standards and samples is competed by a biotinylated CRP sandwiched by the immobilized antibody and streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.
Reagent Preparation Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. EIA Diluent Concentrate (10x): Dilute the EIA Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. CRP Standard: Reconstitute the 25 g of rat CRP Standard with 1.25 ml of EIA Diluent to generate a 20 g/ml of solution. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare triplicate standard points by serially diluting the Standard solution (20 g /ml) 1:4 with EIA Diluent to produce 5, 1.25, 0.313 and 0.078 g/ml . EIA Diluent serves as the zero standard (0 g/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [CRP] (g/ml) 1 part Standard (20 g/ml) P1 20.000 P2 1 part P1 + 3 part EIA Diluent 5.000 P3 1 part P2 + 3 part EIA Diluent 1.250 P4 1 part P3 + 3 part EIA Diluent 0.313 P5 1 part P4 + 3 part EIA Diluent 0.078 P6 EIA Diluent 0.000 Biotinylated Rat CRP (1x): Dilute Biotinylated Rat CRP with 4 ml EIA Diluent to produce a working solution. Allow to sit for 10 minutes with gentle agitation prior to use. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the Wash Buffer Concentrate 1:20 with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with EIA Diluent. Any remaining solution should be frozen at -20°C.
Sample Collection Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10 minutes and assay. Dilute samples 1:400 into EIA Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles (EDTA or Heparin can also be used as anticoagulant). Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. Remove serum and assay. Dilute samples 1:400 into EIA Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles.
Assay Procedure Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 25 µL of standard or sample per well, and immediately add 25 µL of Biotinlylated rat CRP to each well (on top of the Standard or sample) and mix gently. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer. Invert the plate and decant the contents, and hit it 4-5 times on absorbent paper towel to completely remove liquid at each step. Add 50 µL of Streptavidin-Peroxidase Conjugate to each well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash five times with 200 µL of Wash Buffer. Add 50 µL of Chromogen Substrate per well and incubate for about 10 minutes or till the optimal blue color density develops. Gently tap plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. Please note that after the reaction is stopped for about 10 minutes, some black particles may be generated at high concentration point, which will reduce the readings.
Calculation of Results Calculate the mean value of the duplicate or triplicate readings for each standard and sample. To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit. Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.
Assay Precision Intra-assay and inter-assay coefficients of variation were 5.5 % and 7.6% respectively.
Restrictions For Research Use only
Handling Advice The kit should not be used beyond the expiration date.
Storage 4 °C/-20 °C
Storage Comment Store unopened kit at 2-8°C up to expiration date. Opened EIA Diluent may be stored for up to 1 month at 2-8°C. Store reconstituted reagents at -20°C or below. Opened unused strip wells may return to the foil pouch with the desiccant pack, reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.
Product cited in: Ridker, Buring, Shih et al.: "Prospective study of C-reactive protein and the risk of future cardiovascular events among apparently healthy women." in: Circulation, Vol. 98, Issue 8, pp. 731-3, 1998 (PubMed).

Ridker, Cushman, Stampfer et al.: "Plasma concentration of C-reactive protein and risk of developing peripheral vascular disease." in: Circulation, Vol. 97, Issue 5, pp. 425-8, 1998 (PubMed).

Tracy, Lemaitre, Psaty et al.: "Relationship of C-reactive protein to risk of cardiovascular disease in the elderly. Results from the Cardiovascular Health Study and the Rural Health Promotion Project." in: Arteriosclerosis, thrombosis, and vascular biology, Vol. 17, Issue 6, pp. 1121-7, 1997 (PubMed).

Ridker, Cushman, Stampfer et al.: "Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men." in: The New England journal of medicine, Vol. 336, Issue 14, pp. 973-9, 1997 (PubMed).

Kuller, Tracy, Shaten et al.: "Relation of C-reactive protein and coronary heart disease in the MRFIT nested case-control study. Multiple Risk Factor Intervention Trial." in: American journal of epidemiology, Vol. 144, Issue 6, pp. 537-47, 1996 (PubMed).

Liuzzo, Biasucci, Gallimore et al.: "The prognostic value of C-reactive protein and serum amyloid a protein in severe unstable angina." in: The New England journal of medicine, Vol. 331, Issue 7, pp. 417-24, 1994 (PubMed).

Catalog No. ABIN612804

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