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This ELISA kit is of competitive format. Competitive ELISA, also known as inhibition ELISA, is a surface/plate based assay, where the plate is coated with capture antibodies reactive to the molecule of interest. The sample (containing native molecule of interest) and enzyme conjugated recombinant protein (the competing molecule) are added to the coated wells. Since the amount of enzyme conjugated molecule in each well is constant, the level of native molecule in the sample will determine the binding ratio of enzyme conjugated molecule vs. native molecule. After an incubation period, any unbound antibody is washed off. Enzyme substrate (for example, TMB for HRP) is added to each well and will be transformed into a blue precipitate, the amount of which is linearly proportional to the amount of enzyme in the well. The precipitate is then turned into yellow by adding the acid stop solution and the concentration of yellow precipitate is read at 450nm for light absorbance (O.D. value). The O.D. is then used to calculate the amount of molecule of interest in each well, by comparing each sample well against the standard curve. The standard curve is generated using the same principle but instead of adding samples, a series of recombinant molecules with known concentrations are added to 6-8 wells.
| Product Name | Human Cortisol ELISA Kit |
|---|---|
| SKU/Catalog Number | EK7002 |
| Description | Competitive High Sensitivity ELISA kit for Quantitative Detection of Human Cortisol. 96wells/kit, with removable strips. |
| Cite This Product | Human Cortisol ELISA Kit (Boster Biological Technology, Pleasanton CA, USA, Catalog # EK7002) |
| 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 | 20 ng/ml *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 | 20-400 ng/ml *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 |
| Microwells coated with Cortisol MAb | 12x8x1 Microwells |
| Cortisol Standards | 7 vials ( ready to use) 0.5 ml |
| Enzyme Conjugate (20x) | 1 vial 1.2ml |
| Assay Diluent | 1 bottle (ready to use) 24ml |
| TMB Substrate | 1 bottle (ready to use) 12ml |
| Stop Solution | 1 bottle (ready to use) 12ml |
| 20X Wash concentrate | 1 bottle 25ml |
1. Distilled or deionized water
2. Precision pipettes
3. Disposable pipette tips. Multichannel pipettes are recommended in the condition of large amount of samples in the detection.
4. ELISA reader capable of reading absorbance at 450nm
5. Absorbance paper or paper towel
6. Graph paper
Cortisol (hydrocortisone, compound F) is the most potent glucocorticoid synthesized from cholesterol. Cortisol is found in the blood either as free Cortisol, or bound to corticosteroid-binding globulin (CBG). Cortisol production has an ACTH-dependent circadian rhythm with peak levels in the early morning and a nadir at night. The factors controlling this circadian rhythm are not completely defined. Serum levels are highest in the early morning and decrease throughout the day. In the metabolic aspect, Cortisol promotes gluconeogenesis, liver glycogen deposition, and the reduction of glucose utilization. Immunologically, Cortisol functions as an important anti inflammatory, and plays a role in hypersensitivity, immunosuppression, and disease resistance. It has also been shown that plasma Cortisol levels elevate in response to stress. Abnormal Cortisol levels are seen with a variety of different conditions: with adrenal tumors, prostate cancer, depression, and schizophrenia. Elevated Cortisol levels and lack of diurnal variation have been identified in patients with Cushing's disease.
Click the images to enlarge.
1. For Research Use Only. Not for use in diagnostic procedures.
2. 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.
3. Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit reagents are handled.
4. The components in this kit are intended for use as an integral unit. The components of different lots should not be mixed.
5. It is recommended that standards, control and serum samples be run in duplicate.
6. 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.
7. Do not use sodium azide as preservative. Sodium azide inhibits HRP enzyme activities.
1. Collect blood specimens and separate the serum immediately.
2. Typically, specimens may be stored refrigerated at (2°C to 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.
Cortisol-enzyme Conjugate Solution: Dilute the Cortisol enzyme conjugate 1:21 with assay diluent in a suitable container. For example, dilute 100?l of conjugate with 2ml of assay diluent buffer for 10 wells (A slight excess of solution is made).
Wash Buffer: Prepare 1X Wash Buffer by adding the contents of the bottle (25ml, 20X) to 475 ml of distilled or deionized water. Store at room temperature (18-26°C).
Prior to assay, allow reagents to stand at room temperature (20-25°C).
Gently mix all reagents before use.
1. Place the desired number of coated strips into the holder.
2. Pipette 25 ul of Cortisol standards, control and patient’s sera.
3. Add 200 ul of Cortisol Enzyme Conjugate to all wells.
4. Thoroughly mix for 10 seconds.
5. Incubate for 60 minutes at room temperature (20-25°C).
6. Remove liquid from all wells. Wash wells three times with 300 ?l of 1X wash buffer. Blot on absorbent paper towels.
7. Add 100 ul of TMB substrate to all wells.
8. Incubate for 15 minutes at room temperature (20-25°C).
9. Add 50 ul of stop solution to all wells. Shake the plate gently to mix the solution.
10. Read absorbance on ELISA Reader at 450 nm within 20 minutes after adding the stop solution.
The standard curve is constructed as follows:
1. Check Cortisol standard value on each standard vial. This value might vary from lot to lot. Make sure you check the value on every kit. See example of the standard attached.
2. To construct the standard curve, plot the absorbance for Cortisol standards (vertical axis) versus Cortisol 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.
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