**Human Apelin 12 (AP12) ELISA Kit Instruction Manual**
**Kit Specification:**
The Human Apelin 12 (AP12) ELISA Kit is available in 48-well or 96-well configurations. It includes a standard dilution of 1.5 ml × 1 bottle, enzyme standard reagent of 3 ml × 1 bottle (for 48-well) or 6 ml × 1 bottle (for 96-well). This reagent is intended for research use only.
**Calculation Method:**
To determine the concentration of AP12 in the sample, plot the standard curve using the concentration as the x-axis and OD values as the y-axis. The sample's OD value is then compared to the standard curve to estimate its concentration. Alternatively, a linear regression equation can be derived from the standard curve data. Substitute the sample’s OD value into this equation to calculate the actual concentration, and multiply by the dilution factor to obtain the final result.
**Kit Composition:**
- Sealing film: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 0.5 ml × 1 bottle, 2700 ng/L
- Enzyme standard: 1×48 / 1×96
- Sample diluent: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Developer A: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Chromogen B: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Wash solution: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Concentrated wash solution: 20 ml × 20 times (48) / 20 ml × 30 times (96)
**Storage Conditions & Expiration:**
- Storage: 2–8°C
- Shelf Life: 6 months from the date of receipt
**Principle of the Assay:**
This kit employs a double-antibody sandwich ELISA method to quantify human Apelin 12 (AP12) in biological samples. The microtiter plate is pre-coated with purified anti-Apelin 12 antibodies. After adding the sample, AP12 binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming an immune complex. Following washing, TMB substrate is added, which changes color under the catalytic action of HRP. The intensity of the color is directly proportional to the AP12 concentration in the sample. Absorbance is measured at 450 nm, and results are calculated using a standard curve.
**Purpose:**
This kit is designed for the quantitative determination of Apelin 12 in human serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids.
**Sample Preparation Guidelines:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant. Centrifuge after mixing for 10–20 minutes.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via repeated freeze-thaw cycles before centrifugation.
- **Tissue Homogenate:** Weigh the tissue, add PBS, homogenize, centrifuge, and collect the supernatant.
**Important Notes:**
- All samples should be processed promptly. If not tested immediately, store at -20°C, avoiding repeated freeze-thaw cycles.
- Avoid using samples containing NaN₃, as it may inhibit HRP activity.
- Ensure all reagents are equilibrated to room temperature before use.
- Do not reuse sealing films to prevent cross-contamination.
- Handle substrates away from light.
- Strictly follow the protocol to ensure accurate results.
- Treat all waste materials as biohazardous.
- Do not mix reagents from different batches.
- In case of discrepancies, refer to the English manual as the official guide.
**Performance Characteristics:**
- Linearity: Correlation coefficient (R²) ≥ 0.95
- Intra-assay variation < 9%
- Inter-assay variation < 11%
- Detection range: 0.2 IU/L – 6 IU/L
**Operation Steps:**
1. **Standard Dilution:** Prepare serial dilutions of the standard.
2. **Sample Loading:** Add 40 μL of sample diluent and 10 μL of sample to each well.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Wash 5 times with diluted wash buffer.
5. **Enzyme Addition:** Add 50 μL of enzyme-labeled reagent to each well.
6. **Second Incubation:** Incubate for another 30 minutes at 37°C.
7. **Color Development:** Add 50 μL of TMB A and B, incubate at 37°C for 15 minutes.
8. **Stop Reaction:** Add 50 μL of stop solution to terminate the reaction.
9. **Measurement:** Read OD at 450 nm within 15 minutes.
**Technical Support:**
Free technical support is available during working hours. Contact us for assistance with sample testing and experimental optimization.
This kit provides a reliable and efficient method for quantifying Apelin 12 in various biological matrices, ensuring high accuracy and reproducibility in research settings.
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