**Human E2 ELISA Kit – For the quantitative in vitro determination of Human Estradiol concentrations in serum, plasma, celiac fluid, tissue homogenate, and body fluids. FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
Before using this product, please read the entire package insert carefully. This kit is designed for research purposes only and should not be used in diagnostic or therapeutic procedures.
**INTENDED USE AND TEST PRINCIPLE**
This E2 ELISA Kit is intended for laboratory research use only. The test is based on a competitive enzyme immunoassay principle. A stop solution changes the color from blue to yellow, and the intensity of the color correlates with the concentration of estradiol (E2). Calibration standards are run alongside the samples to generate a standard curve, allowing for accurate quantification of E2 levels in the test samples.
**SAMPLE COLLECTION AND STORAGE**
- **Serum**: Use a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation at 2000×g for 20 minutes. Remove the serum and assay immediately, or aliquot and store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma**: Collect using heparin as an anticoagulant. Centrifuge within 30 minutes of collection at 2000×g for 30 minutes at 2–8°C. Store at -20°C. Avoid freeze-thaw cycles.
- **Cell culture supernatants, tissue homogenates, and other biological fluids**: Centrifuge to remove particulates and assay immediately, or aliquot and store at -20°C. Avoid freeze-thaw cycles.
*Note: Ensure adequate centrifugation, and avoid hemolysis or granules in the sample.*
**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 must be stored at 2–8°C. Check the expiration date on the label.
| Name | 96 determinations | 48 determinations |
|-------------------------------|-------------------|-------------------|
| MicroELISA Stripplate | 12×8 strips | 12×4 strips |
| Standard (6 vials, 0.5 ml/vial) | 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: 400, 200, 100, 50, 25, 12.5 pmol/L. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.*
**PRECAUTIONS**
1. Do not mix reagents from different kit lots. Standards, conjugates, and plates are matched for optimal performance. Use only manufacturer-supplied reagents.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use. Do not thaw using water baths.
3. Do not use reagents beyond their expiration date.
4. Use only deionized or distilled water for reagent dilutions.
5. Do not remove microtiter plate strips from the storage bag until needed. Store unused strips at 2–8°C with desiccant.
6. Use fresh disposable pipette tips for each transfer to prevent contamination.
7. Avoid using acid-dissolvable knives, as there is no guaranteed method to ensure safety from infectious agents. All blood derivatives should be treated as potentially infectious.
8. Dispose of all samples following proper inactivation protocols.
9. Liquid waste: Add sodium hypochlorite to achieve a final concentration of 1.0%, let stand for 30 minutes before disposal.
10. Substrate Solution may be easily contaminated. If it appears bluish, do not use.
11. Chromogen Solution B contains 20% acetone; keep away from heat and flame.
12. Allow all reagents to reach room temperature (20–25°C) before starting the assay.
**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. It is recommended to run standards and samples in duplicate.
2. Add 50 µL of standard or sample to the appropriate wells. Blank well receives no addition.
3. Add 100 µL of HRP-conjugate reagent to all wells except the blank. Cover with an adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the microtiter plate 4 times.
- **Manual Washing**: Aspirate contents into a sink or waste container. Fill each well completely with 1X Wash Solution, then aspirate again. Repeat four times. Invert and blot dry.
- **Automated Washing**: Aspirate all wells, wash four times with 1X Wash Buffer. Set fill volume at 350 µL/well. Invert and blot dry.
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 will change from blue to yellow. Read OD at 450 nm within 15 minutes.
**CALCULATION OF RESULTS**
1. Plot the average OD (450 nm) of each standard against its concentration on the Y-axis.
2. Subtract the mean OD of the blank well from all readings.
3. Construct the standard curve using graph paper or software.
4. To determine E2 concentration in samples, locate the OD value on the Y-axis, draw a horizontal line to the curve, then a vertical line to the X-axis to find the corresponding concentration.
5. Results may vary due to operator technique, pipetting, washing, incubation time/temperature, or kit age. Each user should prepare their own standard curve.
6. Intra-assay CV: ≤12.5 pmol/L – 400 pmol/L.
7. Sensitivity: Minimum detectable dose <10 pmol/L.
8. Cross-reactivity: No significant cross-reactivity observed.
9. Storage: Store at 2–8°C for frequent use; for long-term storage, keep at -20°C. Shelf life: 6 months at -20°C.
**NOTES**
Please read all instructions carefully before proceeding. Always follow good laboratory practices and safety protocols.
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