TNNI3 CLIA Kit

Catalog No. ABIN504773

This Chemiluminescent ELISA kit is designed for the quantitative measurement of Human TNNI3.

Quick Overview for TNNI3 CLIA Kit (ABIN504773)

Target: TNNI3 (Cardiac Troponin I (TNNI3))

Reactivity: Human

Detection Method: Chemiluminescent

Method Type: Sandwich ELISA

Application: ELISA

Product Details TNNI3 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 Circulating Troponin-I concentrations in Human Serum 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 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 - 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 Troponin-I 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 tp 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). Incubate for five (5) minutes at room temperature in the dark. 10. Read the relative light units in each well for 0.2 1.0 seconds. The results should be read within thirty (30) minutes of adding the signal reagent solution. NOTE: Always add reagents with multi-channel micropipettes in the same order to minimize reaction time differences between wells.

Restrictions: For Research Use only

Handling

Target Details

Target: TNNI3 (Cardiac Troponin I (TNNI3))

Alternative Name: Troponin I (cTnI)

Background: The cardiac-specific isoforms of Troponin I (cTnI) has been known as a marker of heart damage and myocardial cell death, due to myocardial infarction, for just over 10 years. Troponin-I (cTnI, 24 kDa) is the inhibitory subunit of the Troponin complex of striated muscle. Most of the Troponin (I & T) proteins are located within the contractile apparatus of the striated muscle. The concentration of these subunits is increased in circulation for many days after AMI, because release from the structural elements requires degradation of myofibril itself. This location and release from the specific myocardial tissue and their sustained appearance in circulation make Troponin subunits reliable bio-markers of AMI. Unlike CK-MB and Myoglobin, Troponin does not suffer from non-specificity issues. The immunological determination of cTnI does have some issues surrounding it but those are mainly due to lack of mass standardization, the presence of post-translational modified cTnI in the circulation and variations in the antibody cross-reactivities to the various detectable forms of cTnI. However, cTnI, unlike Myoglobin, has not been found in circulation in marathon runners and other cases of skeletal injury or trauma. Careful selection of antibodies and diligent preparation of stable cTnI for calibration permits cTnI to be an excellent biochemical marker of myocardial damage. Troponin I (cTnI) is the inhibitory subunit of the Troponin complex, which regulates the calcium modulated interaction of actin and myosin in striated muscle. The complex is a heterodimer consisting of troponins C, I and T, which are tightly bound to the contractile apparatus, hence, circulating concentrations are low. Even though Troponin C and T are both considered to be equally good markers for AMI, the NH2 terminus of cTnI has 31 additional amino acids not present in the skeletal isoforms and has generated an interest in easily creating cTnI-specific MoAbs, by researchers. Troponin I, however, has its own problems that researchers are dealing with. Troponin I is not very stable. A major portion of it is bound with troponin C (cTnC) and the remaining small part is free in circulation. Again, the post translational modifications, including selective degradation, covalent complex formation and phosphorylation of cTnI in the post-ischemic myocardium complicate the matters further more. Also, the cTnI proteolysis is even more extensive in human myocardium. Some researchers attribute it in part to the heterogeneity of disease states present in a given patient population. The key to overcoming these problems is a very careful selection of antibodies, the matrix for stabilizing the cTnI and the cTnI protein used as the standard itself. Monobind has been able to successfully put together an assay that promises to have overcome those issues. In this method, Troponin-I 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 Troponin-I are added and the reactants mixed. Reaction between the various Troponin-I antibodies and native Troponin-I forms a sandwich complex that binds with the streptavidin coated to the well. After the completion of the required incubation period, the enzyme- Troponin-I antibody bound conjugate is separated from the unbound enzyme- Troponin-I 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 Troponin-I 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 Troponin-I concentration.