**Rabbit Epidermal Growth Factor (EGF) ELISA Kit Instruction Manual**
**Kit Specifications:**
This Rabbit EGF ELISA Kit is available in 48-well or 96-well configurations. It includes the following components:
- Standard Diluent: 1.5 ml × 1 bottle
- Enzyme Standard Reagent: 3 ml × 1 bottle (for 48-well) or 6 ml × 1 bottle (for 96-well)
- Sealing Film: 2 pieces (for 48-well) or 2 pieces (for 96-well)
- Standard: 0.5 ml × 1 bottle, concentration 2700 ng/L
- Sample Diluent: 3 ml × 1 bottle (for 48-well) or 6 ml × 1 bottle (for 96-well)
- Developer A: 3 ml × 1 bottle (for 48-well) or 6 ml × 1 bottle (for 96-well)
- Chromogen B: 3 ml × 1 bottle (for 48-well) or 6 ml × 1 bottle (for 96-well)
- Stop Solution: 3 ml × 1 bottle (for 48-well) or 6 ml × 1 bottle (for 96-well)
- Concentrated Wash Solution: 20 ml × 20 times (for 48-well) or 20 ml × 30 times (for 96-well)
**Storage Conditions and Shelf Life:**
- Kit Storage: 2–8°C
- Shelf Life: 6 months from the date of manufacture
**Purpose:**
This kit is designed for the quantitative determination of epidermal growth factor (EGF) in rabbit serum, plasma, urine, cell culture supernatants, and tissue homogenates. It is intended for research use only.
**Principle of Operation:**
The kit utilizes a double-antibody sandwich ELISA method. The microplate is pre-coated with a specific antibody against rabbit EGF. After adding the sample and HRP-labeled EGF antibody, an immune complex is formed. Following washing, TMB substrate is added, which turns blue under the action of HRP and then changes to yellow when the reaction is stopped. The intensity of the color is directly proportional to the EGF concentration in the sample. The optical density (OD) is measured at 450 nm, and the EGF concentration is determined by comparing the sample OD value to a standard curve.
**Sample Preparation and Handling:**
1. **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for ~20 minutes. Collect the supernatant; if precipitate forms, re-centrifuge.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge at 2000–3000 rpm for ~20 minutes. Collect the supernatant.
3. **Urine:** Collect in a sterile tube and centrifuge at 2000–3000 rpm for ~20 minutes. Carefully collect the supernatant.
4. **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for ~20 minutes. For intracellular components, lyse cells by repeated freeze-thaw cycles, then centrifuge again.
5. **Tissue Samples:** Weigh the tissue, add PBS (pH 7.4), homogenize, and centrifuge at 2000–3000 rpm for ~20 minutes. Collect the supernatant.
6. **General Notes:** Process samples as soon as possible after collection. If not tested immediately, store at -20°C, avoiding repeated freezing and thawing. Do not use samples containing NaN₃, as it inhibits HRP activity.
**Procedure:**
1. **Standard Dilution and Loading:** Add 100 μL of standard solution to the first and second wells on the plate. Perform serial dilutions (50 μL each) and mix thoroughly.
2. **Sample Addition:** Add 100 μL of diluted sample or blank to the designated wells.
3. **Incubation:** Incubate the plate at 37°C for 1 hour.
4. **Washing:** Remove liquid, wash 3–5 times with wash buffer.
5. **Add Developer A and B:** Add 100 μL of Developer A, incubate for 15 minutes, then add 100 μL of Chromogen B.
6. **Stop Reaction:** Add 100 μL of Stop Solution.
7. **Read OD Value:** Measure absorbance at 450 nm using a microplate reader.
8. **Data Analysis:** Plot the standard curve using standard concentrations vs. OD values. Calculate sample concentrations using linear regression or interpolation. Multiply by the dilution factor if applicable.
**Service Commitment:**
- Delivery within the agreed time after payment.
- Free technical support during working hours.
- Free sample testing services available upon request to ensure optimal results.
**Note:** Always follow the manufacturer’s instructions and perform proper quality control checks.
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