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C-Reactive Protein (CRP) ELISA Kit

Details for Product No. ABIN649050, Supplier: Login to see
  • crp
  • PTX1
  • AI255847
  • Aa1249
  • Ab1-341
  • Ab2-196
  • Ac1-114
  • Ac1262
  • Ac2-069
  • Ba2-693
  • APCS
  • 0610010I23Rik
  • AW743261
  • C77570
  • CRP2
  • CRP4
  • Crp
  • ESP1
  • Hlp
  • CRP
  • GRP-CB
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Purpose Immunoenzymometric assay (TYPE 3): The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (enzyme and immobilized), with different and distinct epitope recognition, in excess, and native antigen. In this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monoclonal anti-CRP antibody. Upon mixing monoclonal biotinylated antibody, the enzyme-labeled antibody and a serum containing the native antigen, reaction results between the native antigen and the antibodies, without competition or steric hindrance, to form a soluble sandwich complex. After equilibrium is attained, the antibodybound fraction is separated from unbound antigen by decantation or aspiration. The enzyme activity in the antibodybound fraction is directly proportional to the native antigen concentration. By utilizing several different serum references of known antigen values, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained.
Detection Method Colorimetric
Components A. CRP Calibrators (1ml/vial). Six vials of references CRP Antigen at levels of 0 (A), 0. 5 (B), 2. 0 (C), 5. 0 (D), 15 (E) and 30 (F) µg/ml. Store at 2-8°C. A preservative has been added. Note: The calibrators, human serum based, were calibrated using a reference preparation, which was assayed against the international reference material CRM 470. B. CRP Enzyme Reagent (13ml/vial):. One vial containing Biotin labeled monoclonal mouse IgG and Anti-CRP HRP in buffer, dye, and preservative. Store at 2-8°C. C. Streptavidin Coated Plate (96 wells). One 96-well microplate coated with streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C. D. Serum Diluent (20ml). One vial of serum diluent containing buffer salts and a dye. Store at 2-8°C. E. Wash Solution Concentrate (20 ml). One vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. F. Substrate A (7ml/vial). One bottle containing tetramethylbenzidine (TMB) in buffer. Store at 2-8°C. G. Substrate B (7ml/vial). One bottle containing hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. H. Stop Solution (8m/vial). One bottle containing a strong acid (1N HCl). Store at 2-30°C. I. Product Instructions: Note 1: Do not use reagents beyond the kit expiration date. Note 2: Opened reagents are stable for 60 days when stored at 2-8°C. Note 3: Above reagents are for a single 96-well microplate.
Material not included 1. Pipette capable of delivering 25µl & 50µl volumes with a precision of better than 1. 5%. 2. Dispenser(s) for repetitive deliveries of 0. 100ml and 0. 350ml volumes with a precision of better than 1. 5%. 3. Microplate washers or a squeeze bottle (optional). 4. Microplate Reader with 450nm and 620nm wavelength absorbance capability. 5. Absorbent Paper for blotting the microplate wells. 6. Plastic wrap or microplate cover for incubation steps. 7. Vacuum aspirator (optional) for wash steps. 8. Timer. 9. Quality control materials.
Background Summary and Explanation of the test: C-Reactive Protein has traditionally been used to diagnose and monitor acute inflammation. It was named as such for its ability to bind and precipitate the C-polysacchride of pneumococcus. It is an alpha globulin (MW 110-140 kD). CRP is synthesized in the liver and is normally present as a trace constituent of serum or plasma at levels less than 0. 3 mg/dl. It has numerous physiological functions similar to those of immunoglobulins and acts as a host defense mechanism. CRP is one of the acute phase proteins, the circulatory levels of which rise during general, non-specific response to a wide variety of diseases. These include infections by bacteria, acute phase of rheumatoid arthritis, abdominal abscesses and inflammation of the bile duct. High levels of CRP may also be found in patients with some viral infections, tuberculosis, acute infectious hepatitis, many other necrotic and inflammatory disease, burn and surgical trauma victims. Although the elevated levels of CRP are not indicative of any particular disease, the sudden rise of CRP does indicate an inflammatory process. CRP levels rise in circulation within 24-48 hours following acute tissue damage, reach a peak (upto 1000 times the constitutive level) and decrease with the resolution of trauma or inflammation. The elevated levels of CRP may last for several days before reaching back to normal levels. Since, elevated levels of CRP are always associated with pathological changes, the CRP assays provide useful information for the diagnosis and therapeutic monitoring of inflammatory processes and associated diseases. Measurement of CRP by high sensitivity CRP assays adds to the predictive value of other cardiac markers like Myoglobin, CK-MB, cTnI and cTnT to assess the risk of cardiovascular and peripheral vascular disease. Rifai and Ridker - in a study for CDC - have proposed that medical decision points established by prospective epidemiological studies be used to interpret individual patient CRP results in risk assessment for cardiovascular disease. This is similar to the approach used by the National Cholesterol Education Program for blood lipids that requires that assays for CRP be standardized to provide comparable results. With the advent of sensitive methodologies like Elisa the use of high sensitivity CRP assays is becoming more routine to aid in the determination of inflammation due to cardiovascular trauma. Since CRP is not specific for anything in particular, hs-CRP assay results should be used in conjunction with other historical, physiological and pathological findings. In this method, CRP calibrator, patient specimen or control is first added to a streptavidin coated well. Biotinylated monoclonal and enzyme labeled antibodies (directed against distinct and different epitopes of CRP) are added and the reactants mixed. Reaction between the various CRP antibodies and native CRP forms a sandwich complex that binds with the streptavidin coated to the well. After the completion of the required incubation period, the enzyme-CRP antibody bound conjugate is separated from the unbound enzyme-CRP conjugate by aspiration or decantation. The activity of the enzyme present on the surface of the well is quantitated by reaction with a suitable substrate to produce color. The employment of several serum references of known CRP levels permits construction of a dose response curve of activity and concentration. From comparison to the dose response curve, an unknown specimen's activity can be correlated with CRP concentration. Intended Use: The Quantitative Determination of C -Reactive protein (CRP) concentration in Human Serum, or Plasma by a Microplate Immunoenzymometric assay. Q. C. Parameters: Maximum Absorbance (Calibrator F) equal greater than1. 3Maximum Absorbance (Calibrator A) equal lower than 0. 1
Research Area Serum/Plasma Proteins, Inflammation
Application Notes Precautions: For In Vitro Diagnostic Use. Not for Internal or External Use in Humans or Animals. All products that contain human serum have been found to be non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV Antibodies by FDA licensed reagents. Since no known test can offer complete assurance that infectious agents are absent, all human serum products should be handled as potentially hazardous and capable of transmitting disease. Good laboratory procedures for handling blood products can be found in the Center for Disease Control / National Institute of Health, Biosafety in Microbiological and Biomedical Laboratories, 2nd Edition, 1988, HHS Publication No. (CDC) 88-8395.
Reagent Preparation 1. Serum Diluent: Dilute the serum diluent to 200ml in a suitable container with distilled or deionized water. Store at 2-8°C. 2. Wash Buffer: Dilute contents of wash solution to 1000 ml with distilled or deionized water in a suitable storage container. Store at room temperature 20-27 °C for up to 60 days. 3. Working Substrate Solution: Pour the contents of the amber vial labeled Solution A into the clear vial labeled Solution B. Place the yellow cap on the clear vial for easy identification. Mix and label accordingly. Store at 2 - 8 °C. Note: Do not use the working substrate if it looks blue. 4. Patient Sample Dilution (1/200): Dispense 0. 010ml (10µl) of each patient specimen into 2ml of serum diluent. Cover and vortex or mix thoroughly by inversion. Store at 2-8°C for up to 48 hours. Note: THE CALIBRATORS ARE READY TO USE.
Sample Collection The specimens shall be blood, serum or plasma in type and the usual precautions in the collection of venipuncture samples should be observed. For accurate comparison to established normal values, a fasting morning serum sample should be obtained. The blood should be collected in a plain redtop venipuncture tube without additives or anti-coagulants (for serum) or evacuated tube(s) containing EDTA or heparin. Allow the blood to clot for serum samples. Centrifuge the specimen to separate the serum or plasma from the cells. Samples may be refrigerated at 2-8°C for a maximum period of five days. If the specimen(s) cannot be assayed within this time, the sample(s) may be stored at temperatures of -20 °C for up to 30 days. Avoid repetitive freezing and thawing. When assayed in duplicate, 0. 050ml of the diluted specimen is required.
Calculation of Results A dose response curve is used to ascertain the concentration of C-reactive protein in unknown specimens. 1. Record the absorbance obtained from the printout of the microplate reader. 2. Plot the absorbance for each duplicate serum reference versus the corresponding CRP concentration in µg/ml on linear graph paper (do not average the duplicates of the serum references before plotting). 3. Draw the best-fit curve through the plotted points. 4. To determine the concentration of CRP for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration (in mIU/ml) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated). Note: If the sample values need to be represented in mg% divide the value obtained (in StepNumber4) by 10 to convert the values in mg/dl (or mg %).
Restrictions For Research Use only
Handling Advice Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20-27°C). 1. Format the microplates' wells for each serum reference, control and patient specimen to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8°C. 2. Pipette 0. 025 ml (25µl) of the appropriate serum reference, diluted control or specimen (see Patient Sample Preparation above) into the assigned wells. 3. Add 0. 100 ml (100µl) of the CRP Enzyme Reagent to each well. It is very important to dispense all reagents close to the bottom of the coated well. Note: Use a multichannel pipet to quickly dispense the Enzyme Reagent to avoid drift if the dispensing is to take more than a few minutes. 4. Swirl the microplate gently for 20-30 seconds to mix and cover. 5. Incubate 15 minutes at room temperature. Discard the contents of the microplate by decantation or aspiration. If decanting, tap and blot the plate dry with absorbent paper. 7. Add 350µl of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two additional times for a total of three washes. An automatic or manual plate washer can be used. Follow the manufacturer's instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and Repeat two additional times. 8. Add 0. 100 ml (100µl) of working substrate solution to all wells (see Reagent Preparation Section). DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION. 9. Incubate at room temperature for 15 minutes. 10. Add 0. 050ml (50µl) of stop solution to each well and mix gently for 15-20 seconds. 11. Read the absorbance in each well at 450nm (using a reference wavelength of 620-630nm to minimize well imperfections) in a microplate reader. The results should be read within 30 minutes of adding the stop solution. Note: Always add reagents in the same order to minimizereaction time differences between wells.
Storage 4 °C/-20 °C
Background publications Kimberly, Vesper, Caudill et al.: "Standardization of immunoassays for measurement of high-sensitivity C-reactive protein. Phase I: evaluation of secondary reference materials." in: Clinical chemistry, Vol. 49, Issue 4, pp. 611-6, 2003 (PubMed).

Yudkin, Stehouwer, Emeis et al.: "C-reactive protein in healthy subjects: associations with obesity, insulin resistance, and endothelial dysfunction: a potential role for cytokines originating from adipose tissue?" in: Arteriosclerosis, thrombosis, and vascular biology, Vol. 19, Issue 4, pp. 972-8, 1999 (PubMed).

Ridker, Rifai, Pfeffer et al.: "Inflammation, pravastatin, and the risk of coronary events after myocardial infarction in patients with average cholesterol levels. Cholesterol and Recurrent Events (CARE) Investigators." in: Circulation, Vol. 98, Issue 9, pp. 839-44, 1998 (PubMed).

Ridker, Glynn, Hennekens: "C-reactive protein adds to the predictive value of total and HDL cholesterol in determining risk of first myocardial infarction." in: Circulation, Vol. 97, Issue 20, pp. 2007-11, 1998 (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).

Macy, Hayes, Tracy: "Variability in the measurement of C-reactive protein in healthy subjects: implications for reference intervals and epidemiological applications." in: Clinical chemistry, Vol. 43, Issue 1, pp. 52-8, 1997 (PubMed).

Kushner, Rzewnicki: "The acute phase response: general aspects." in: Baillière's clinical rheumatology, Vol. 8, Issue 3, pp. 513-30, 1994 (PubMed).