TNNI3 ELISA Kit

Catalog No. ABIN649060

Product Details TNNI3 ELISA Kit

Target: TNNI3 (Cardiac Troponin I (TNNI3))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

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. The interaction is illustrated by the following equation: kaEnzAb(m)+ AgcTnI + BtnAb(m) EnzAb(m)-Ag(cTnI)-BtnAb(m) k-aBtnAb(m) equal Biotinylated Monoclonal Antibody (Excess Quantity)AgcTnI. equal Native Antigen (Variable Quantity)EnzAb(m)equal Enzyme labeled MoAb (Excess Quantity)EnzAb(m)-AgcTnI. -BtnAb(m) equal AntigenAntibodies complexka equal Rate Constant of Associationka equal Rate Constant of DissociationSimultaneously, the complex is deposited to the well through the high affinity reaction of streptavidin and biotinylated antibody. This interaction is illustrated below: EnzAb (m)-AgcTnI. -BtnAb (m)+StreptCW(immobilized complexStrepC. W. equal Streptavidin immobilized on wellImmobilized complex equal sandwich complex bound to the solid surfaceAfter sufficient time for reaction, 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.

Analytical Method: Quantitative

Components: A. Troponin-I Calibrators (1. 0 ml/vial) (Lyophilized). Six vials of references for Troponin-I antigen at levels of 0 (A), 1. 0 (B), 3. 0 (C), 6. 0 (D), 15 (E), and 30 (F) ng/ml. Reconstitute each vial with 1. 0ml of distilled or deionized water. The reconstituted calibrators are stable for 24 hours at 2-8°C. In order to store for a longer period of time aliquot the reconstituted calibrators in cryo vials and store at -20 °C. DO NOT FREEZE THAW MORE THAN ONCE. A preservative has been added. Note: The calibrators, human serum based, were calibrated using NIST standard for cTnI Number 2921. B. Troponin-I Enzyme Conjugate (13 ml/vial). One vial containing enzyme labeled affinity purified antibody and biotin labeled monoclonal mouse IgG in buffer, dye, and preservative. Store at 2-8°C. C. Streptavidin Microplate(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. Wash Solution (Concentrate) (20 ml). One vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. E. Substrate A (7. 0ml/vial). One bottle containing tetramethylbenzidine (TMB) in buffer. Store at 2-8°C. F. Substrate B (7. 0ml/vial). One bottle containing hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. G. Stop Solution (8. 0ml/vial). One bottle containing a strong acid (1N HCl). Store at 2-30°C. H. Product Instructions.

Material not included: 1. Pipette (s) capable of delivering 25µl and 100µl 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% (optional). 3. Microplate washer 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. Storage container for storage of wash buffer. 10. Distilled or deionized water. 11. Quality Control Materials.

Application Details

Application Notes: Precautions: 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.

Plate: Pre-coated

Reagent Preparation:

1. Wash Buffer: Dilute contents of wash solution to 1000ml with distilled or deionized water in a suitable storage container. Store at room temperature 20-27 °C for up to 60 days. 2. 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.

Sample Collection: 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 2-8°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. 050 ml of the specimen is required.

Calculation of Results:

A dose response curve is used to ascertain the concentration of Troponin I in unknown specimens. 1. Record the absorbance obtained from the printout of the microplate reader as outlined in Example 1 2. Plot the absorbance for each duplicate serum reference versus the corresponding cTnI concentration in ng/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 cTnI 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 ng/ml) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated).

Restrictions: For Research Use only

Handling

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 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 (25µl) of the appropriate calibrators, controls and samples into the assigned wellsAdd 0. 100 ml (100µl) of the Troponin-I Enzyme Reagent to each well. It is very important to dispense all reagents close to the bottom of the microwell. 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. Swirl the microplate gently for 20-30 seconds to mix. Cover with a plastic wrap. Incubate for 15 minutes at room temperature (20-27°C). Discard the contents of the microplate by decantation or aspiration. If decanting, tap and blot the plate dry with absorbent paper. 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 used, fill each well to the top by squeezing the container. Avoiding air bubbles. 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. 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 minimize reaction time differences between wells.

Storage: 4 °C/-20 °C

Target Details for TNNI3

Target: TNNI3 (Cardiac Troponin I (TNNI3))

Alternative Name: Troponin I (cTnI) (TNNI3 Products)

Synonyms: CMD1FF ELISA Kit, CMD2A ELISA Kit, CMH7 ELISA Kit, RCM1 ELISA Kit, TNNC1 ELISA Kit, cTnI ELISA Kit, CMD1Z ELISA Kit, CMH13 ELISA Kit, TN-C ELISA Kit, TNC ELISA Kit, TNNC ELISA Kit, Tn1 ELISA Kit, TnIc ELISA Kit, ctnIc ELISA Kit, XTnIc ELISA Kit, c-troponin ELISA Kit, cTNT ELISA Kit, cmh7 ELISA Kit, ctni ELISA Kit, tnnc1 ELISA Kit, TNNI3 ELISA Kit, TnI ELISA Kit, cTNI ELISA Kit, AI874626 ELISA Kit, TnC ELISA Kit, cTnC ELISA Kit, tncc ELISA Kit, Tncc ELISA Kit, troponin I3, cardiac type ELISA Kit, troponin C1, slow skeletal and cardiac type ELISA Kit, troponin I, cardiac 3 ELISA Kit, troponin I type 3 (cardiac) ELISA Kit, troponin I3, cardiac type S homeolog ELISA Kit, troponin I3, cardiac type L homeolog ELISA Kit, cardiac troponin I ELISA Kit, troponin C, cardiac/slow skeletal ELISA Kit, TNNI3 ELISA Kit, TNNC1 ELISA Kit, Tnni3 ELISA Kit, tnni3.S ELISA Kit, tnni3.L ELISA Kit, tnni3 ELISA Kit, LOC100462680 ELISA Kit, Tnnc1 ELISA Kit

Background: Summary and Explanation of the test: The cardiac-specific isoform 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-translationally 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 MoAb's, 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 tropinin 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. 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 color. 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. Intended Use: The Quantitative Determination of Circulating Troponin-I concentrations in Human Serum by a Microplate Immunoenzymometric assay. Q. C. Parameters: In order for the assay results to be considered valid the following criteria should be met: 1. The absorbance (OD) of calibrator A should be lower than 0. 07. 2. The absorbance (OD) of calibrators F should be greater than 1. 3. 3. Four out of six quality control pools should be within the established ranges.