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This Bosterbio ELISA kit is designed to measure the concentration of FT4 in human serum by Competitive ELISA (Enzyme-Linked Immunosorbent Assay). The polystyrene micro-well plate in this kit has been pre-coated with an anti-Human FT4 antibody. Sample or standards are added to the wells along with a fixed quantity of biotinylated FT4 and incubated. The FT4 found in the sample or standards competes with the biotinylated FT4 for limited binding sites on the immobilized anti-Human FT4 antibody. Excess unbound biotinylated FT4 and sample or standard FT4 is washed from the plate. Avidin-HRP conjugate is added, incubated and washed. An enzymatic reaction is then produced through the addition of substrate which is catalyzed by the immobilized HRP to generate a blue color product that changes yellow after adding acidic stop solution. The density of yellow coloration is measured by reading the absorbance at 450 nm which is quantitatively proportional to the amount of biotinylated FT4 captured in the well and inversely proportional to the amount of FT4 which was contained in the sample or standard.
| Product Name | Human Free Thyroxine (FT4) ELISA Kit (Competitive ELISA) |
|---|---|
| SKU/Catalog Number | EK7054 |
| Description | Competitive High Sensitivity ELISA kit for Quantitative Detection of Human FT4. 96wells/kit, with removable strips. |
| Cite This Product | Human Free Thyroxine (FT4) ELISA Kit (Competitive ELISA) (Boster Biological Technology, Pleasanton CA, USA, Catalog # EK7054) |
| Validated Species | Human |
| Application | ELISA *Our Boster Guarantee covers the use of this product in the above tested applications. |
| Cross Reactivity | There is no detectable cross-reactivity. |
| Pack Size | 96wells/kit, with removable strips. |
| Sensitivity | 4.0 pmol/L *Sensitivity, or Lower Limit of Detection (LLD), is the minimum level of target protein the ELISA assay can detect. We measure 20 blank wells and if the O.D. value is 2 standard deviations higher than the blanks' average O.D. the sample can be deemed positive. |
|---|---|
| Assay Range | 4-64 pmol/L *This assay range is determined using common samples. For samples with low target protein concentrations, users can adjust the standard curve to extend the lower limit of assay range. |
| Sample Type | Serum *The above listed samples are the ones valided with the assay. If you do not see your sample of interest listed, as long as there is enough level of target protein present in the sample, this Picokine? ELISA kit should detect it. **For protocol and tips regarding preparing your sample of interest, please check our ELISA sample preparation guide. |
| Storage | Store the kit at 2°C to 8°C. Keep microwells sealed in a dry bag with desiccants. The reagents are stable until expiration of the kit. Do not expose reagent to heat, sun, or strong light. Avoid multiple freeze-thaw cycles(Shipped with wet ice.) |
| Description | Quantity | Volume | Buffers |
| Pre-coated 96-well strip microplate | 1 | 8 strips of 12 wells | Rabbit Anti- FT4 Antibody, Polystyrene micro-well plate |
| Human FT4 Standards(S1~S5) | 5 | 1ml | FT4 (4, 8, 16, 32, 64 pmol/L), 0.02M PBS, 50% detoxification serum, 0.1% Proclin-300 |
| Avidin-HRP conjugate | 1 | 6ml | Avidin-HRP conjugate, 0.02M PBS, 20% new-born calf serum, 0.01% azophloxine, 0.1% Proclin-300 |
| Biotinylated antigen | 1 | 6ml | Biotinylated FT4 analogue, 0.02M PBS, 20% new-born calf serum, 0.1% Proclin-300 |
| Controls | 2 | 1ml | T4, 100% natural protein, 0.1% Proclin-300 |
| 20X Wash Buffer Concentrate | 1 | 15ml | 0.2M PBS containing 0.5% tween 20 |
| Color Developing Reagent A | 1 | 7ml | 11m mol/L Urea hydrogen peroxide |
| Color Developing Reagent B | 1 | 7ml | 2m mol/L 3,3'5,5’-Tetramethylbenzidine |
| Stop Solution | 1 | 7ml | 2mol/L Sulphuric acid |
| Plate Sealers | 2 | Piece |
1. Microplate Reader capable of reading absorbance at 450nm.
2. Automated plate washer (optional)
3. Pipettes and pipette tips capable of precisely dispensing 0.5 μl through 1 ml volumes of aqueous solutions. Multichannel pipettes are recommended for large amount of samples.
4. Deionized or distilledwater.
5. 500ml graduated cylinders.
6. Test tubes for dilution.
Click the images to enlarge.
Human FT4 ELISA Kit standard curve
1. For Research Use Only. Not for use in diagnostic procedures.
2. For laboratory use.
3. Potential biohazardous materials: The calibrator and controls contain human source components, which have been tested and found non-reactive for hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However, there is no test method that can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent. These reagents should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control/National Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories" 1984.
4. Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit reagents are handled.
5. The components in this kit are intended for use as an integral unit. The components of different lots should not be mixed.
6. It is recommended that standards, control and serum samples be run in duplicate.
7. Optimal results will be obtained by strict adherence to this protocol. Accurate and precise pipetting, as well as following the exact time and temperature requirements prescribed are essential. Any deviation from this may yield invalid data.
1. Collect blood specimens and separate the serum immediately.
2. Typically, specimens may be stored refrigerated at (2-8. C) for 5 days. If storage time exceeds 5 days, store frozen at (-20. C) for up to one month.
3. Avoid multiple freeze-thaw cycles.
4. Prior to assay, frozen sera should be completely thawed and mixed well.
5. Do not use grossly lipemic specimens.
Prepare 1X Wash buffer by adding the contents of the bottle (25 ml, 20X) to 475 ml of distilled or deionized water. Store at room temperature.
Bring all specimens and kit reagents to room temperature (20-25°C) and gently mix.
1. Format the microplates wells for control, standard and patient samples to be assayed in duplicate. Place any unused microwell strips back into the aluminum bag, seal and store at 2-8°C.
2. Pipette 25ul of fT4 standards, control and samples into the assigned well.
3. Add 50ul of fT4 enzyme conjugate to all wells.
4. Add 50ul of Anti-T4 Biotin Solution to all the wells.
5. Swirl the microplate gently for 20-30 seconds to mix the reagents.
6. Incubate for 60 minutes at room temperature (18-26. C).
7. Remove liquid from all wells. Fill wells with 300ul 1X wash buffer (see buffer preparation above) Wash three times. Blot on absorbent paper towels.
8. Add 100ul of TMB substrate to all wells.
9. Incubate for 15 minutes at room temperature.
10. Add 50ul of stop solution to all wells. Shake the plate gently to mix the solution.
11. Read absorbance on ELISA Reader at 450 nm within 15 minutes after adding the stopping solution.
The standard curve is constructed as follows:
1. Check fT4 standard value on each standard vial. This value might vary from lot to lot. Make sure you check the value on every kit.
2. To construct the standard curve, plot the absorbance for the Ferritin standards (vertical axis) versus versus fT4 standard concentrations (horizontal axis) on a linear graph paper. Draw the best curve through the points.
3. Read the absorbance for controls and each unknown sample from the curve. Record the value for each control or unknown sample.
1. Do not use sodium azide as preservative. Sodium azide inhibits HRP enzyme activities.
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