**Human E2 ELISA Kit – For the quantitative in vitro determination of Human Estradiol concentrations in serum, plasma, celiac fluid, tissue homogenate, and body fluids. Intended for laboratory research use only. NOT FOR USE IN DIAGNOSTIC PROCEDURES. Please read this entire package insert before using the product.**
**INTENDED USE AND TEST PRINCIPLE**
This E2 ELISA kit is specifically designed for research purposes in a laboratory setting and is not intended for diagnostic or therapeutic use. The test is based on the principle of enzyme-linked immunosorbent assay (ELISA), where the concentration of estradiol (E2) in the samples is quantified by comparing the optical density (OD) readings to a standard curve generated from known E2 concentrations.
The Stop Solution changes the color from blue to yellow, and the intensity of the color correlates with the E2 concentration. Calibration standards are run alongside the samples, allowing the operator to create a standard curve by plotting OD values against E2 concentrations. The E2 levels in the test samples are then determined by comparing their OD values to the standard curve.
**SAMPLE COLLECTION AND STORAGE**
- **Serum**: Use a serum separator tube. Allow the sample to clot for 2 hours at room temperature or overnight at 4°C. Centrifuge for 20 minutes at approximately 2000×g. 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 repeated freeze-thaw cycles.
- **Cell culture supernatants, tissue homogenates, and other biological fluids**: Centrifuge to remove particulates. Assay immediately or aliquot and store at -20°C. Avoid repeated freeze-thaw cycles.
*Note: Ensure adequate centrifugation and avoid hemolysis or granules in the samples.*
**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 should be stored at 2–8°C. Check the expiration date on the label.
| Reagent | 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 are 400, 200, 100, 50, 25, 12.5 pmol/L.*
*If sample values exceed the highest standard, dilute the sample with Sample Diluent and repeat the assay.*
**PRECAUTIONS**
1. Do not mix reagents from different kit lots. Standards, conjugate, and microtiter plates are matched for optimal performance. Only use reagents provided by the manufacturer.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use. Do not thaw samples or reagents in a water bath.
3. Do not use reagents past their expiration date.
4. Use only deionized or distilled water when diluting reagents.
5. Do not open microtiter plate strips until ready to use. Store unused strips in their pouch with desiccant at 2–8°C.
6. Use fresh pipette tips for each transfer to prevent contamination.
7. Do not use microtiter plates from storage bags until needed.
8. All blood-derived products should be considered potentially infectious. Follow good laboratory practices.
9. Dispose of all samples properly to inactivate viruses.
10. Liquid waste: Add sodium hypochlorite to achieve a final concentration of 1.0%. Let stand for at least 30 minutes before disposal.
11. Substrate solution may become contaminated. If it appears bluish, do not use.
12. Chromogen B contains 20% acetone. Keep away from heat or flame.
13. Allow all reagents to reach room temperature 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 beginning. It is recommended to add standards and samples in duplicate to the microtiter strip plate.
2. Add 50 µl of standard or sample to the appropriate wells. The 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 the contents into a waste container. Fill each well completely with 1X Wash Solution, then aspirate again. Repeat four times. Invert the plate and blot dry.
- **Automated Washing:** Aspirate all wells, then wash four times with 1X Wash Buffer. Set fill volume at 350 µL/well/wash. 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 the OD at 450 nm within 15 minutes using a microplate reader.
**CALCULATION OF RESULTS**
1. Generate a standard curve by plotting the average OD (450 nm) of each standard concentration on the Y-axis versus the corresponding E2 concentration on the X-axis.
2. Subtract the blank OD value from all other readings before interpretation.
3. Construct the standard curve using graph paper or statistical software.
4. To determine the E2 concentration in each sample, locate the OD value on the Y-axis, draw a horizontal line to the curve, then a vertical line to the X-axis to read the E2 concentration.
5. Variations in technique, timing, or reagent age may affect results. Each user should generate their own standard curve.
6. Intra-assay CV (%) and inter-assay range: 12.5 pmol/L to 400 pmol/L.
7. Sensitivity: Minimum detectable dose of Human E2 is typically less than 10 pmol/L.
8. No significant cross-reactivity or interference was observed.
9. Storage: Store at 2–8°C for frequent use; store at -20°C for long-term (up to six months).
10. Always follow safety protocols and dispose of waste properly.
**NOTES**
- This kit is intended for research only.
- Ensure proper training and handling of all reagents.
- Maintain accurate records of all procedures and results.
- Consult the user manual for detailed instructions and troubleshooting.
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