Myoglobin CLIA Kit

Catalog No. ABIN504772

Human Myoglobin ELISA Kit, Chemiluminescent assay for quantification of Human Myoglobin.

Quick Overview for Myoglobin CLIA Kit (ABIN504772)

Target: Myoglobin (MB)

Reactivity: Human

Detection Method: Chemiluminescent

Method Type: Sandwich ELISA

Application: ELISA

Product Details Myoglobin 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-myoglobin 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 Myoglobin concentrations in Human Serum, or Urine 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 red-top venipuncture tube without additives. Allow the blood to clot. Centrifuge the specimen to separate the serum from the cells. Centrifuging serum samples before a complete clot forms may result in the presence of fibrin. To prevent erroneous results due to the presence of fibrin make sure that complete clot formation has taken place prior to the centrifugation of the samples. Urine samples may be collected without preservatives and stored at 2-8C for up to 8 hours. For longer storage the samples should be immediately aliquoted and stored at -20C. (Please contact Monobind for special Urinary Myoglobin procedure). Samples may be refrigerated at 2_x001E_8C for a maximum period of two (2) days. If the specimen(s) cannot be assayed within this time, the sample(s) may be stored at temperatures of _x001E_20C for up to 30 days. Avoid repetitive freezing and thawing. When assayed in duplicate, 0.050ml 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. 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 - 27( 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 Myoglobin 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. 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). Incubate at room temperature for five (5) minutes in the dark. Read the Relative Light Units (RLU) in each well using a 96 well 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: Myoglobin (MB)

Alternative Name: Myoglobin

Background: Myoglobin is an enzyme, found primarily in cardiac and skeletal muscle. It is an oxygen biding protein and exists as a monomeric form of hemoglobin. Being a monomer of hemoglobin which is a tetramer - myoglobin has one fourth the molecular weight (18 kD) of hemoglobin. Since Myoglobin, like FABP (fatty acid binding protein) is a low molecular mass ctyoplasmic protein present not only in heart but also in the skeletal muscle it is difficult to use it as a plasma marker for muscle cell viability to discriminate between heart or skeletal muscle injury. The Myoglobin content of human heart, however, is lower than that of skeletal muscle. Serial measurement of biochemical markers is now accepted universally as an important determinant in ruling in or ruling out acute myocardial infarction. Myoglobin is one of the most important myocardial markers used in ruling out acute myocardial infarction (AMI) within 2h of admission because of chest pains. AMI disrupts cardiac cell membranes, releasing intracellular cardiac proteins into the vascular system. Some of the proteins including myoglobin, creatine kinase-MB (CK-MB), lactate dehydrogenase type-1 (LD1) and cardiac troponin subunits I and T (TnI & TnT) have proven useful in diagnosing AMI. The optimal clinical utility of each marker depends on specific protein characteristics. Myoglobin, being the smallest of these markers, diffuses rapidly throughout the vascular system and provides the earliest indication of AMI. Myoglobin levels rise between 0.5 2.0 hours after the onset of chest pains and peak within 5-12 hours. The kidneys rapidly eliminate myoglobin from the system, restoring normal circulating concentrations within 16-36 hours. Since the protein rapidly clears from the system, myoglobin concentrations can reliably indicate reinfarction. Also, myoglobin measurements can preclude AMI. According to the Heart Emergency Room (ER) Program model two consecutive low measurements, the first upon admission of the patient and the second two hours later, negatively predict AMI in nearly 100% of the cases. Myoglobin participates in aerobic metabolism in cardiac and skeletal muscle cells thus showing high concentrations in muscle traumas. Renal failures and other kidney problems exhibit high levels of myoglobin levels as well. Most complications are accompanied by distinct clinical symptoms that make the differential diagnosis possible. Serial testing for myoglobin and CK-MB isoenzyme mass on presentation and 3, 6 and 9 h later in patients with symptoms suggestive of acute ischemic coronary syndrome presenting with a non-diagnostic or equivalent electrocardiogram has been shown to be more effective than continuous serial electrocardiograms, echocardiography, and graded exercise testing. (Monobind has an excellent Chemi test system Cat# 2975-300 for the determination of circulating levels of CK-MB in human serum or plasma). In this method, Myoglobin 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 Myoglobin are added and the reactants mixed. Reaction between the various Myoglobin antibodies and native Myoglobin forms a sandwich complex that binds with the streptavidin coated to the well. After the completion of the required incubation period, the enzyme- Myoglobin antibody bound conjugate is separated from the unbound enzyme-Myoglobin 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 (Myoglobin) 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 Myoglobin concentration.

Pathways: Brown Fat Cell Differentiation