Troponin I Elisa Test Kit High Accuracy 96wells / kit Quantitative Measurement

Troponin I (TNNI3) ELISA Kit 96wells/kit, high accuracy, Elisa Sandwich method, for quantitative measurement
 
 
Product Name:  Troponin I ELISA Kit
 
Intended Use:
 
Troponin I (TNNI3) ELISA Kit is an enzyme-linked immunosorbent assay for measuring human troponin I in serum, plasma, and cell culture media.
 
Introduction:
 
Troponin is an important contractile regulatory protein of skeletal and cardiac muscle. Troponin exists as a heteromeric protein complex (Figure 1) in striated muscle and consists of three subunits: troponin C (TnC), troponin T (TnT), and troponin I (TnI). Each of these subunits plays a specific regulatory function in this complex. TnC binds Ca2+ ions, TnT binds tropomyosin, and TnI binds actin; when complexed, they attach to the actin filament. Introduction
 
Troponin I exists in 3 isoforms. The TnI isoform found in the myocardium, cTnI, has been shown to be a powerful diagnostic marker for assessing heart disorders. Following a heart attack (myocardial infarction), damaged cells release cTnI into the blood; these elevated levels can be seen 3-6 hours postinfarction and remain elevated for several days. Over the last 20 years, cTnI has emerged as the preferred biomarker for myocardial infarction diagnosis and is considered more sensitive/specific than other diagnostic targets such as CK-MB, total CK, myoglobin, or LDH.

Figure 1: The Troponin Complex
 
Cell Biolabs’ Human Cardiac Troponin I ELISA Kit is an enzyme immunoassay developed for detection and quantitation of the human cardiac Troponin I protein. The kit has a detection sensitivity limit of 50 pg/mL cTnI. Each kit provides sufficient reagents to perform up to 96 assays including standard curve and Troponin I samples.
 
 
Principle of Test:

An anti-Troponin I coating antibody is adsorbed onto a microtiter plate. Troponin I protein present in the sample or standard binds to the antibodies adsorbed on the plate; a biotinylated anti-Troponin I antibody is added and binds to the antigen captured by the first antibody. Following incubation and wash steps, a streptavidin-enzyme conjugate is added and binds to the biotinylated anti-Troponin I antibody. Unbound streptavidin-enzyme conjugate is removed during a wash step, and substrate solution is added to the wells. A colored product is formed in proportion to the amount of Troponin I present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450 nm. A standard curve is prepared from purified cardiac Troponin I and sample concentration is then determined
 
Kit Components
 

1.  Microtiterwells, 12 x 8 (break apart) strips, 96 wells; Wells coated with Treponema pallidum antigen. (incl. 1 strip holder and 1 cover foil)
2.  Pos. Control ***, 1 vial, 1.0 mL, ready to use; colored yellow, red cap.
3.  Neg. Control ***, 1 vial, 2.0 mL, ready to use; colored yellow, yellow cap.
4.  Cut-off Control ***, 1 vial, 1.0 mL, ready to use; colored yellow, black cap.
5.   Enzyme Conjugate **, 1 vial, 13 mL, ready to use,
6. Substrate Solution, 1 vial, 14 mL, ready to use, Tetramethylbenzidine (TMB).
7.  Stop Solution, 1 vial, 14 mL, ready to use, contains 0.2 mol/l H2SO4, Avoid contact with the stop solution. It may cause skin irritations and burns.
8.  Wash Solution *, 1 vial, 30 mL (20X concentrated for 600 mL), pH 7.2 ± 0.2 see „Preparation of Reagents“.

* contains 0.03 % ProClin 300
** contains 0.03 % ProClin 300 + 0.01 % Gentamicin sulphate
*** contains 0.03 % ProClin 300 + 0.015 % 5-bromo-5-nitro-1,3-dioxane (BND) + 0.010 % 2-methyl-2H-isothiazol-3-one (MIT)
 
 
ASSAY PROCEDURE
 
 

1. Bring all reagents and samples to room temperature (18 - 25ºC) before use. It is recommended that all standards and samples be run at least in duplicate.

2. Add 100µL of each standard (see Reagent Preparation step 3) and sample into appropriate wells. Cover wells and incubate for 2.5 hours at room temperature with gentle shaking.

Note: Overnight incubations at 4°C with gentle shaking can be performed, but may increase overall signals including background.

3. Discard the solution and wash 4 times with 1X Wash Buffer. Wash by filling each well with Wash Buffer (300µL) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.

4. Add 100µL of 1X prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.

5. Discard the solution. Repeat the wash as in step 3.

6. Add 100µL of prepared Streptavidin-HRP solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.

7. Discard the solution. Repeat the wash as in step 3.

8. Add 100µL of TMB Substrate to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.

9. Add 50 µl of Stop Solution to each well.

10. The plate must be evaluated within 30 minutes of stopping the reaction. Measure absorbance on an ELISA plate reader set at 450nm and 550nm. Subtract 550nm values from 450nm values to correct for optical imperfections in the microplate. If 550nm is not available, measure absorbance at 450nm only. Omitting the 550nm measurement will result in higher absorbance values.

Performance Characteristics
 
Typical Data: These standard curves are for demonstration only. A standard curve must be run with each assay.
 
Sensitivity: 100 pg/mL
 
The sensitivity or Lower Limit of Detection (LLD) was determined by assaying replicates of zero and the standard curve. The mean signal of zero + 2 standard deviations read in dose from the standard curve is the LLD. This value is the smallest dose that is not zero with 95% confidence.
 
Spiking & Recovery: Pooled serum, plasma, and cell culture media samples were spiked with recombinant Human Troponin I. Endogenous Human Troponin I levels were determined by testing non-spiked samples alongside spiked aliquots of the same samples. Expected values were calculated by adding endogenous Troponin I levels to those of the spiked control. Percent recovery was calculated by dividing observed by expected values.
 

 
 
Linearity: The serum, plasma, and cell culture media samples were spiked with recombinant Human Troponin I, serially diluted in sample diluent and evaluated. Observed values were compared to expected values to calculate percent recovery and demonstrate the dilution linearity of the assay.
 

]