**Human Apelin 12 (AP12) ELISA Kit Instruction Manual**
**Kit Specifications:**
- **Configuration:** 48-well or 96-well format
- **Standard Dilution:** 1.5 mL × 1 vial
- **Enzyme Standard Reagent:** 3 mL × 1 vial (for 48-well), 6 mL × 1 vial (for 96-well)
- **Storage Conditions:** 2–8°C
- **Expiration Date:** 6 months from the date of receipt
**Principle of Operation:**
This ELISA kit utilizes a sandwich immunoassay method to quantify human apelin 12 (AP12) in biological samples. The microplate is pre-coated with a specific antibody against AP12. After adding the sample and enzyme-labeled detection antibody, a complex is formed. TMB substrate is then added, producing a color change that is proportional to the AP12 concentration. The reaction is stopped, and the absorbance is measured at 450 nm. A standard curve is plotted using known concentrations, and the sample concentration is calculated accordingly.
**Kit Components:**
- Sealing Film: 2 pieces (48-well), 2 pieces (96-well)
- Standard: 2700 ng/L, 0.5 mL × 1 vial
- Enzyme Standard: 1×48 or 1×96, stored at 2–8°C
- Sample Diluent: 3 mL × 1 vial (48), 6 mL × 1 vial (96), stored at 2–8°C
- Developer A: 3 mL × 1 vial (48), 6 mL × 1 vial (96), stored at 2–8°C
- Chromogen B: 3 mL × 1 vial (48), 6 mL × 1 vial (96), stored at -8°C
- Wash Buffer: 3 mL × 1 vial (48), 6 mL × 1 vial (96), stored at 2–8°C
- Concentrated Wash Solution: (20 mL × 20 times) × 1 bottle or (20 mL × 30 times) × 1 bottle, stored at 2–8°C
**Sample Preparation and Requirements:**
1. **Serum:** Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant, mix well, centrifuge, and collect supernatant.
3. **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
4. **Cell Culture Supernatant:** Centrifuge, discard pellet, and use supernatant. For intracellular components, lyse cells by freezing and thawing.
5. **Tissue:** Homogenize in PBS, centrifuge, and collect supernatant.
6. **Storage:** Store samples at -20°C if not tested immediately. Avoid repeated freeze-thaw cycles.
7. **Note:** NaN3-containing samples are not suitable for this assay due to HRP inhibition.
**Operating Steps:**
1. **Standard Dilution:** Prepare a serial dilution of the standard and load into designated wells.
2. **Sample Loading:** Add 40 μL of sample diluent followed by 10 μL of sample (final dilution 5x).
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Wash 5 times with diluted wash buffer.
5. **Add Enzyme Conjugate:** Add 50 μL of enzyme reagent to each well.
6. **Second Incubation:** Repeat step 3.
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.
9. **Measurement:** Read OD at 450 nm within 15 minutes.
**Notes:**
- Allow the kit to equilibrate at room temperature before use.
- Avoid cross-contamination by using a new sealing film for each test.
- Ensure accurate pipetting and maintain consistent timing.
- Always run a standard curve and consider sample dilution if OD values exceed the range.
- Handle all reagents carefully and dispose of waste as biohazardous material.
- Do not mix reagents from different batches.
- Follow the manual strictly; results should be based on microplate reader readings.
**Performance Characteristics:**
- **Linearity:** Correlation coefficient (R) ≥ 0.95
- **Intra-batch CV < 9%, Inter-batch CV < 11%
- **Detection Range:** 0.2 IU/L – 6 IU/L
**Purpose:**
This ELISA kit is designed for the quantitative determination of human apelin 12 (AP12) in serum, plasma, urine, cell culture supernatants, and tissue homogenates.
**Service Commitment:**
- Free technical support during working hours.
- Sample testing services available upon request.
- Delivery within the agreed time after payment.
**Storage:**
Store the kit at 2–8°C. Do not freeze.
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