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**Chicken ACTH ELISA Kit – For the quantitative in vitro determination of Chicken adrenocorticotropic hormone (ACTH) concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
Before using this product, please read this entire package insert carefully. This kit is designed for research purposes only and should not be used in diagnostic or therapeutic applications.
**INTENDED USE AND TEST PRINCIPLE**
The Chicken ACTH ELISA Kit is intended for laboratory research use only. It utilizes a competitive enzyme-linked immunosorbent assay (ELISA) to quantitatively measure ACTH levels in various biological samples. The color change from blue to yellow, induced by the Stop Solution, allows for optical density (OD) measurement at 450 nm. Standard curves are generated alongside sample measurements, enabling accurate determination of ACTH concentration in unknown samples.
**STORAGE AND HANDLING OF SAMPLES**
- Store all samples at -20°C. Avoid repeated freeze-thaw cycles.
- For cell culture supernatants, tissue homogenates, and other biological fluids: Centrifuge to remove particulates and analyze immediately, or aliquot and store at -20°C.
- Ensure samples are adequately centrifuged, with no hemolysis or visible particles present.
**MATERIALS REQUIRED BUT NOT SUPPLIED**
1. Incubator set at 37°C
2. Microplate reader capable of measuring absorbance at 450 nm
3. Precision pipettes, disposable tips, and absorbent paper
4. Distilled or deionized water
**REAGENTS PROVIDED**
All reagents are stored at 2–8°C. Check the expiration date on the label before use.
| Name | 96 Determinations | 48 Determinations |
|------|-------------------|-------------------|
| Microplate (12×8 strips) | 1 | 1 |
| Standard (6 vials) | 0.5 ml/vial | 0.5 ml/vial |
| Sample Diluent | 6.0 ml | 3.0 ml |
| HRP-Conjugate Reagent | 10.0 ml | 5.0 ml |
| 20X Wash Solution | 25 ml | 15 ml |
| Chromogen Solution A | 6.0 ml | 3.0 ml |
| Chromogen Solution B | 6.0 ml | 3.0 ml |
| Stop Solution | 6.0 ml | 3.0 ml |
| Closure Plate Membrane | 2 | 2 |
| User Manual | 1 | 1 |
| Sealed Bags | 1 | 1 |
*Note: Standard concentrations are 80, 40, 20, 10, 5, and 2.5 pg/mL. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.*
**PRECAUTIONS**
1. Do not mix reagents from different kits. Use only manufacturer-supplied components.
2. Allow all reagents and samples to reach room temperature (20–25°C) before use. Avoid using water baths for thawing.
3. Do not use reagents past their expiration date.
4. Only use deionized or distilled water for dilutions.
5. Keep microtiter plates in sealed bags until ready to use. Unused strips should be stored at 2–8°C with desiccant.
6. Use fresh pipette tips for each transfer to prevent contamination.
7. Wear disposable gloves during the procedure. All blood-derived products should be considered potentially infectious.
8. Dispose of all samples and waste properly.
9. Liquid waste must be treated with sodium hypochlorite at a final concentration of 1.0% for at least 30 minutes before disposal.
10. Substrate solutions are sensitive; avoid contamination. Solution B contains 20% acetone—keep away from heat or flame.
11. Remove all reagents from the refrigerator and allow them to warm to room temperature before use.
**REAGENT PREPARATION AND STORAGE**
**Wash Solution (1X):** Dilute 1 volume of 20X Wash Solution with 19 volumes of deionized or distilled water. Store at 2–8°C for up to one month.
**ASSAY PROCEDURE**
1. Prepare all reagents before starting the assay.
2. Add 50 µL of standard or sample to appropriate wells (excluding blank). Cover with adhesive strip and incubate for 60 minutes at 37°C.
3. Wash the microtiter plate 4 times.
- **Manual washing:** Aspirate contents, fill with 1X Wash Solution, and aspirate again. Repeat 4 times.
- **Automated washing:** Aspirate and wash 4 times with 1X Maintain Buffer. Adjust brush to remove as much liquid as possible, and fill 350 µL per well.
4. After final wash, invert and blot dry with absorbent paper.
5. Add 50 µL of Chromogen Solution A and 50 µL of Chromogen Solution B to each well. Mix gently and incubate for 15 minutes at 37°C, protecting from light.
6. Add 50 µL of Stop Solution to each well. The color should turn yellow. If green or uneven, gently tap the plate.
7. Measure OD at 450 nm within 15 minutes using a microplate reader.
**CALCULATION OF RESULTS**
1. Plot average OD values of standards against their concentrations on a graph.
2. Calculate mean OD for each standard and sample. Subtract blank OD before interpretation.
3. Locate the OD value on the Y-axis, draw a horizontal line to the curve, then a vertical line to the X-axis to determine ACTH concentration.
4. Each user should generate their own standard curve due to potential variations in technique or conditions.
5. Intra-assay and inter-assay CV% are less than 15%.
6. Assay range: 2.5 pg/mL to 80 pg/mL.
7. Sensitivity: <1.0 pg/mL.
8. Cross-reactivity: No significant cross-reactivity observed.
9. Storage: 2–8°C (frequent use); 6 months at -20°C.
**NOTES**
This kit is suitable for researchers working with avian models. Always follow safety protocols and proper waste disposal procedures. For any questions, contact the manufacturer’s technical support team.
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