Creatine Kinase MB Isoenzyme Type II CLIA Kit

Catalog No. ABIN504771

The Human Creatine Kinase MB Isoenzyme Type II ELISA Kit (ABIN504771) is a Chemiluminescent ELISA Kit designed to quantify Human Creatine Kinase MB Isoenzyme Type II.

Quick Overview for Creatine Kinase MB Isoenzyme Type II CLIA Kit (ABIN504771)

Target: Creatine Kinase MB Isoenzyme Type II

Reactivity: Human

Detection Method: Chemiluminescent

Method Type: Sandwich ELISA

Application: ELISA

Product Details Creatine Kinase MB Isoenzyme Type II 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-CK-MB 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 Circulating Creatinine Kinase (MB-Isoform) Concentrations in Human Serum by a Microplate Immunoenzymometric 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 specimen is required.

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. _x000E_ 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.

Test Procedure:

Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20 - 25( C). Format the microplates wells for calibrator, 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. Pipette 0.025 ml (25l) of the appropriate calibrators, controls and samples into the assigned wells. Add 0.100 ml (100l) of the CK-MB Tracer Reagent to each well. It is very important to dispense all reagents close to the bottom of the microwell. Note: Use a multichannel pipette to quickly dispense Enzyme Reagent to avoid drift if the dispensing is to take more than a few minutes. Swirl the microplate gently for 20-30 seconds to mix. Cover with a plastic wrap. Incubate for 15 minutes at room temperature (20-25C). Discard the contents of the microplate by decantation or aspiration. If decanting, tap and blot the plate dry with absorbent paper. 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 used, fill each well to the top by squeezing the container. Avoiding air bubbles. Decant the wash and repeat four (4) additional times. Add 0.100 ml (100l) of working signal reagent to all wells (see Reagent Preparation Section). 9. Read the Relative Light Units (RLUs) 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: Creatine Kinase MB Isoenzyme Type II

Alternative Name: CK-MB (CK-2)

Background: Creatinine kinase (CK) is an enzyme, found primarily in muscle and brain tissue, which exists as three dimeric isoenzymes CKMM (CK-3), CK-MB (CK-2), and CK-BB (CK-1) built from subunits designated M and B. The CK-MB isoenzyme, which has a molecular mass of approximately 87,000 daltons, accounts for 5 to 50% of total CK activity in myocardium. In skeletal muscle, by contrast, it normally accounts for just 1% or less, CK-MM being the dominant form, though the percentage can be as high as 10% in conditions reflecting skeletal muscle injury and regeneration (e.g. severe exercise, muscular dystrophy, polymyositis).2 Serial measurement of biochemical markers is now accepted universally as an important determinant in ruling in or ruling out acute myocardial infarction. CK-MB is one of the most important myocardial markers (in spite of not being cardiac-specific), with well established roles in confirming acute myocardial infarction (AMI) and in monitoring reperfusion during thrombolytic therapy following AMI.2 In AMI, plasma CK-MB typically raises some 3 to 8 hours after the onset of chest pains, peaks within 9 to 30 hours, and returns to baseline levels within 48 to 72 hours7. The pattern of serial CK-MB determinations is more informative than a single determination. One CK-MB measurement, even when taken at an appropriate time, cannot definitively confirm or rule out the occurrence of AMI. High levels might reflect skeletal injury rather than myocardial damage. A value within the reference range might be significant if it represents an increase from the patients baseline levels. Accordingly it has been recommended that CK-MB be measured on admission to the emergency room, and at regulated intervals thereafter. The model described by the Heart Emergency Room (ER) Program (13) documented that serial testing for CK-MB isoenzyme mass on presentation and 3,6 and 9 hours later in patients with symptoms suggestive of acute ischemic coronary syndrome presenting with a non-diagnostic or equivalent electrocardiogram was more effective (100% sensitivity with 100% negative predictive value) than continuous serial electrocardiograms, electrocardiography and graded exercise testing. In this method, CK-MB 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 CK-MB are added and the reactants mixed. Reaction between the various CK-MB antibodies and native CK-MB forms a sandwich complex that binds with the streptavidin coated to the well. After the completion of the required incubation period, the enzyme-CK-MB antibody bound conjugate is separated from the unbound enzyme-CK-MB 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 (CK-MB) levels permits the 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 CK-MB concentration.