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The ACTH Immunoassay is a two-site ELISA for the measurement of the biologically active 39 amino acid chain of ACTH. One antibody is prepared to bind only the C-terminal ACTH 34-39 and this antibody is biotinylated. The other antibody is prepared to bind only the mid-region and N-terminal ACTH 1-24 and this antibody is labeled with HRP for detection. In this assay, calibrators, controls, or patient samples are simultaneously incubated with the enzyme labeled antibody and a biotin coupled antibody in a streptavidincoated microplate well. At the end of the assay incubation, the microwell is washed to remove unbound components and the enzyme bound to the solid phase is incubated with the TMB substrate. Stop solution is then added to stop the reaction and converts the color to yellow. The intensity of the yellow color is directly proportional to the concentration of ACTH in the sample. A dose response curve of absorbance unit vs. concentration is generated using results obtained from the calibrators. Concentrations of ACTH present in the controls and patient samples are determined directly from this curve.
| Product Name | Human ACTH ELISA Kit |
|---|---|
| SKU/Catalog Number | EK7007 |
| Description | A two-site ELISA kit for Quantitative Detection of Human Adrenocorticotropic Hormone (ACTH). 96wells/kit, with removable strips. |
| Cite This Product | Human ACTH ELISA Kit (Boster Biological Technology, Pleasanton CA, USA, Catalog # EK7007) |
| 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 | 6.8 pg/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 | 6.8-531 pg/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 | EDTA plasma *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 Streptavidin | 12x8x1 Microwells |
| Biotinylated ACTH Antibody (Reagent 1) | 2.7 ml |
| Peroxidase (Enzyme) labeled ACTH Antibody | (1 Vial) 2.7 ml |
| Wash Concentrate | (1 Vial) 30 ml |
| TMB Substrate | 1 vial 15ml |
| Stop Solution | 1 Vial 20 ml |
| Calibrators | 5 Vials 2 ml |
| Zero Calibrator | (1 Vial) 4 ml |
| Controls 1 & 2 (CTRL) | (2 Vials) 2 ml |
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
ACTH is a 39-amino acid peptide hormone (MW=4500) secreted by the pituitary to regulate the production of steroid hormones by the adrenal cortex. ACTH increases the synthesis and release of all adrenal sterioids, aldosterone, cortisol and adrenal androgens. It is the principal modulator of cortisol, the most important glucocorticoid in man. As the cortisol level in blood increases, release of ACTH is inhibited directly at the pituitary level. Through this same mechanism, decreasing cortisol levels lead to elevated ACTH levels. In healthy individuals, ACTH reaches a peak in the early morning (6:00 - 8:00 hour) and levels become lowest late in the day and near the beginning of the sleep period. Stress may also override the diurnal variation. Plasma ACTH assays are useful in the differential diagnosis of pituitary Cushing’s disease, Addison’s disease, autonomous ACTH producing pituitary tumors (e.g. Nelson’s syndrome), hypopituitarism with ACTH deficiency and ectopic ACTH syndrome. Primary adrenocortical insufficiencies, Addison’s disease. Hypopituitarism with ACTH deficiency, which is secondary adrenocortical insufficiency, is characterized by low plasma ACTH and cortisol concentrations, and a subnormal, but usually distinct adrenal response to stimulation with synthetic ACTH (Cortrosyn).
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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. This product contains components preserved with sodium azide. Sodium azide may react with lead and copper plumbing to form explosive metal azide. On disposal, flush with a large volume of water.
1. The determination of ACTH should be performed on EDTA plasma.
2. To assay the specimen in duplicate, 400 ul of EDTA plasma is required.
3. Collect whole blood in a lavender [EDTA] tube.
4. The plasma should be promptly separated, preferably in a refrigerated centrifuge, and stored at -20°C or lower.
5. EDTA plasma samples may be stored up to 8 hours at 2-8°C.
6. EDTA plasma samples frozen at -20°C are stable for up to 4 months.
Store all kit components at 2-8°C except Wash Concentrate and Stop Solution.
All reagents except the non-zero calibrators, kit controls and the Wash Concentrate are ready-to-use. Store all reagents at 2-8°C, except the Wash Concentrate, which should be kept at room temperature until dilution to avoid precipitation.
For each of the non-zero calibrators (Calibrator B through F) and kit controls 1 and 2, reconstitute each vial with 2 ml of distilled or deionized water and mix. Allow the vial to stand for 10 minutes and then mix thoroughly by gentle inversion to insure complete reconstitution. Use the calibrators and controls as soon as possible upon reconstitution. Freeze (-20°C) the remaining calibrators and controls as soon as possible after use. Calibrators and controls are stable at -20°C for 6 weeks after reconstitution with up to 3 freeze thaw cycles when handled as recommended in “Procedural Notes” section.
ELISA Reagent A: Wash Concentrate: Mix contents of wash concentrate thoroughly. If precipitate is present in the Wash Concentrate due to storage at lower temperature such as 4°C, dissolve by placing the vial in a 37°C water bath or oven with swirling or stirring. Add wash concentrate (30 ml) to 570 ml of distilled or deionized water and mix. The diluted working wash solution is stable for 90 days when stored at room temperature.
Prior to assay, allow reagents to stand at room temperature (20-25°C).
Gently mix all reagents before use.
1. Place sufficient Streptavidin Coated Strips in a holder to run all six (6) ACTH calibrators, A - F of the ACTH CALIBRATORS (concentration is stated on the vial label), Quality Control Plasma and patient samples.
2. Pipet 200 μl of sample into the designated or mapped well. Freeze (-20°C) the remaining calibrators and controls as soon as possible after use.
3. Add or dispense 25 μl of Reagent 1 (Biotinylated Antibody) into each of the wells which already contain the sample.
4. Add or dispense 25 μl of Reagent 2 (Enzyme Labeled Antibody) into each of the same wells. Cover the microplate(s) with aluminum foil or a tray to avoid exposure to light, and place it on an orbital shaker or rotator set at 170 + 10 rpm for 4 hours + 30 minutes at room temperature (20-25°C).
5. First aspirate the fluid completely and then wash/aspirate each well five (5) times with the Working Wash Solution (prepared from Reagent A), using an automatic microplate washer. The wash solution volume should be set to dispense 0.35 ml into each well.
6. Add or dispense 150 μL of the ELISA Reagent B (TMB Substrate) into each of the wells.
7. With appropriate cover to avoid light exposure, place the microplate(s) on an orbital shaker or rotator set at 170 + 10 rpm for 30 +5 minutes at room temperature (20-25°C).
8. Add or dispense 100 μl of the Stopping Solution into each of the wells. Mix gently.
9. Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to 450 nm against 250 μl of
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