CRP CLIA Kit

Catalog No. ABIN504770

This Chemiluminescent ELISA kit is designed for the quantitative measurement of Human CRP.

Quick Overview for CRP CLIA Kit (ABIN504770)

Target: CRP (C-Reactive Protein (CRP))

Reactivity: Human

Detection Method: Chemiluminescent

Method Type: Sandwich ELISA

Application: ELISA

Product Details CRP CLIA Kit

Purpose: Immunoenzymometric assay (TYPE 3): The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (enzyme conjugated 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-Troponin-I antibody. Upon mixing biotin labeled monoclonal 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.

Analytical Method: Quantitative

Characteristics: The Quantitative Determination of CRP C-Reactive Protein concentration in Human Serum, Plasma or Whole Blood by a Microplate Immunoenzymometric Chemiluminescence assay

Application Details

Application Notes: 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 required tests. 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.

Plate: Pre-coated

Protocol:

Specimien Collection and Preparation:

The specimens shall be blood serum in type and the usual precautions in the collection of venipuncture samples should be observed. The blood should be collected in a plain redtop venipuncture tube without additives or gel barrier. Allow the blood to clot. Centrifuge the specimen to separate the serum from the cells. Samples may be refrigerated at 2_x001E_8(C for a maximum period of five (5) days. If the specimen(s) cannot be assayed within this time, the sample(s) may be stored at temperatures of _x001E_20(C for up to 30 days. Avoid repetitive freezing and thawing. When assayed in duplicate, 0.050 ml of the diluted specimen is required. _x000E_

Reagent Preparation:

1. Wash Buffer Dilute contents of Wash Concentrate to 1000ml with distilled or deionized water in a suitable storage container. Store diluted buffer at room temperature 20-27(C. 2. Working Signal Reagent Solution - Store at 2 - 8(C. Determine the amount of reagent needed and prepare by mixing equal portions of Signal Reagent A and Signal Reagent B in a clean container. For example, add 1 ml of A and 1ml of B per two (2) eight well strips (A slight excess of solution is made). Discard the unused portion if not used within 36 hours after mixing. If complete utilization of the reagents is anticipated, within the above time constraint, pour the contents of Signal Reagent B into Signal Reagent A and label accordingly. 3. Serum Diluent Dilute the serum diluent concentrate to 200ml in a suitable container with distilled or deionized water. Store at 2-8(C. 4. Patient Sample Dilution (1/200) Dispense 0.010ml (10l) 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 forty-eight (48) hours. Note: THE CALIBRATORS ARE READY TO USE.

Test Procedure:

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 (25l) of the appropriate serum reference, diluted specimen or control (see Patient Sample Preparation above) into the assigned wells. 3. Add 0.100 ml (100l) of the CRP Tracer 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 Tracer 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 350l of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat four (4) additional times for a total of five (5) washes. An automatic or manual plate washer can be used. Follow the manufacturers 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 four (4) additional times. 8. Add 0.100 ml (100l) of Working Signal Reagent to all wells (see Reagent Preparation Section). 9. Incubate at room temperature for 5 minutes in the dark. 9. Read the Relative Light Units (RLU) in each well using a microplate luminometer. The results should be read within thirty (30) minutes of adding the Working Signal Reagent. Note: Always add reagents in the same order to minimize reaction time differences between wells.

Restrictions: For Research Use only

Handling

Target Details

Target: CRP (C-Reactive Protein (CRP))

Background: 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-polysaccharide 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, 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, Monobind 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 light. 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.

Pathways: Carbohydrate Homeostasis