TFF3 ELISA Kit

Catalog No. ABIN956208

Product Details TFF3 ELISA Kit

Target: TFF3 (Trefoil Factor 3 (Intestinal) (TFF3))

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: This TFF3 ELISA kit is intended for laboratory research use only and is not for use in diagnostic or therapeutic procedures.

Analytical Method: Quantitative

Characteristics: This TFF3 enzyme linked immunosorbent assay applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific for TFF3. Calibrators or samples are then added to the microtiter plate wells and TFF3 if present will bind to the antibody pre-coated wells. In order to quantitatively determine the amount of TFF3 present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated polyclonal antibody, specific for TFF3 are added to each well to sandwich the TFF3 immobilized on the plate. The microtiter plate undergoes incubation, and then the wells are thoroughly washed to remove all unbound components. Next, A and B substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain TFF3 and enzyme-conjugate antibody will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm.

Components: Microtiter Plate: 96 wells
Calibrator 1 (0 ng/mL)
Calibrator 2 (1 ng/mL)
Calibrator 3 (2.5 ng/mL)
Calibrator 4 (5 ng/mL)
Calibrator 5 (10 ng/mL)
Calibrator 6 (20 ng/mL)
Enzyme Conjugate (1 x 6 mL)
Substrate A (1 x 6 mL)
Substrate B (1 x 6 mL)
Stop Solution (1 x 6 mL)
Wash Buffer (100X concentrate) (1 x 6 mL)
Lysis Buffer Solution (1 x 6 mL)

Material not included: 1. Microplate reader capable of measuring absorbance at 450 nm.
2. Precision pipettes to deliver 2 mL to 1 mL volumes.
3. Adjustable 10-100 mL pipettes for reagent preparation.
4. 100 mL and 1 liter graduated cylinders.
5. Calibrated adjustable precision pipettes, preferably with disposable plastic tips. (A manifold multi- channel pipette is desirable for large assays.)
6. 37°C incubator.
7. Absorbent paper.
8. Distilled or de-ionized water
9. Data analysis and graphing software. Graph paper: linear (Cartesian), log-log or semi-log, or log-logit as desired.
10. Tubes to prepare calibrator or sample dilutions.

Application Details

Plate: Pre-coated

Reagent Preparation:

Bring all kit components and samples to room temperature (18-25°C) before use. Wash Solution Dilute 10 mL of Wash Solution concentrate (100X) with 990 mL of de-ionized or distilled water to prepare 1,000 mL of Wash Solution (1X).

Assay Procedure:

It is recommended that all Calibrators and Samples be added in duplicate to the Microtiter Plate.
1. Secure the desired number of coated wells in the holder, then add 50 µL of Calibrators or Samples to the appropriate well of the antibody pre-coated Microtiter Plate.
2. Add 100 µL of Conjugate to each well. Mix well. Complete mixing in this step is important. Cover and incubate for 1 hour at 37°C.
3. Wash the microtiter plate using one of the specified methods indicated below:
4. Manual Washing: Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well completely with wash solution, then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure four more times for a total of five washes. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly when washing the plate to assure that all strips remain securely in frame.
5. Automated Washing: Aspirate all wells, then wash plate five times using wash solution. Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350 µL/well/wash (range: 350-400 µL). After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. It is recommended that the washer be set for a soaking time of 10 seconds or shaking time of 5 seconds between washes.
6. Add 50 µL substrate A and 50 µL substrate B to each well. Cover and incubate for 10 minutes at 20- 25°C. (Avoid exposure to light.)
7. Add 50 µL stop solution to each well. Mix well.
8. Read the optical density (OD) at 450 nm using a microtiter plate reader immediately.

Calculation of Results:

1. This calibration curve is used to determine the amount in an unknown sample. The calibration curve is generated by plotting the average OD (450 nm) obtained for each of the six calibrator concentrations on the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis.
   2. First, calculate the mean OD value for each calibrator and sample. All OD values are subtracted by the mean value of the zero calibrator before result interpretation. Construct the calibration curve using graph paper or statistical software.
   3. To determine the amount in each sample, first locate the OD value on the Y-axis and extend a horizontal line to the calibration curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration.
   4. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in the result. Each user should obtain their own calibration curve.
   5. The sensitivity by this assay is 0.01 ng/mL.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: All reagents provided are stored at 4°C. Refer to the expiration date on the label.

Expiry Date: The expiry date is stated on the label.

Target Details for TFF3

Target: TFF3 (Trefoil Factor 3 (Intestinal) (TFF3))

Alternative Name: Trefoil Factor 3 (TFF3) (Intestinal) (TFF3 Products)

Synonyms: TFF3 ELISA Kit, ITF ELISA Kit, P1B ELISA Kit, TFI ELISA Kit, mITF ELISA Kit, trefoil factor 3 ELISA Kit, trefoil factor 3, intestinal ELISA Kit, TFF3 ELISA Kit, Tff3 ELISA Kit