**Human Chromogranin A (CgA) ELISA Kit Instruction Manual**
**Kit Specification:**
- 48-well or 96-well configuration
- Standard Diluent: 1.5 mL × 1 bottle
- Enzyme Standard Reagent: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
- **Note:** This reagent is for research use only.
**Standard Curve Preparation:**
To determine the concentration of CgA in samples, plot the standard concentrations (on the x-axis) against their corresponding OD values (on the y-axis). The standard curve can be drawn on graph paper or using software. Once the curve is established, the sample’s OD value is compared to the curve to find its corresponding concentration. Multiply this by the dilution factor to obtain the actual sample concentration. Alternatively, calculate the linear regression equation from the standard data and plug in the sample’s OD value to compute the final concentration.
**Kit Components:**
- Sealing Film: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 2700 ng/L, 0.5 mL × 1 bottle
- Enzyme Standard: 1×48 or 1×96, stored at 2–8°C
- Sample Diluent: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well), stored at 2–8°C
- Developer A: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well), stored at 2–8°C
- Chromogen B: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well), stored at -20°C
- Stop Solution: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well), stored at 2–8°C
- Concentrated Wash Solution: 20 mL × 20 times or 20 mL × 30 times, stored at 2–8°C
**Principle of Operation:**
This kit uses a double-antibody sandwich ELISA method to detect human chromogranin A (CgA). A microplate coated with purified anti-CgA antibody is used as the solid phase. After adding the sample, CgA binds to the immobilized antibody. An HRP-labeled anti-CgA antibody is then added, forming a complex. After washing, TMB substrate is introduced, which turns blue under HRP catalysis and becomes yellow upon acid addition. The intensity of the color is directly proportional to the CgA concentration. The absorbance is measured at 450 nm, and the concentration is calculated based on the standard curve.
**Purpose:**
The kit is designed to quantify CgA levels in human serum, plasma, urine, cerebrospinal fluid, ascites, pleural effusion, and cell culture supernatants.
**Service Commitment:**
- Delivery period: From payment to delivery
- Free technical support during working hours
- Free proxy testing services available upon request
- Storage conditions: 2–8°C, shelf life: 6 months
**Sample Preparation and Handling:**
- **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.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant.
- **Urine:** Collect in a sterile container, centrifuge at 2000–3000 rpm for 20 minutes. Discard any precipitate.
- **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via freezing-thawing cycles, then centrifuge again.
- **Tissue Samples:** Homogenize in PBS (pH 7.4), centrifuge, and collect supernatant. Store at 2–8°C after thawing.
- **Storage:** Process samples immediately after collection. If not tested right away, store at -20°C, avoiding repeated freeze-thaw cycles.
- **Avoid NaN3**, as it inhibits HRP activity.
**Operating Procedure:**
1. **Standard Dilution & Loading:** Prepare serial dilutions of the standard. Add 100 μL to the first two wells, then perform a 1:2 dilution across the plate.
2. **Sample Loading:** Add 40 μL of sample diluent and 10 μL of sample to each test well. Ensure no contact with the well walls.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Dilute concentrated wash solution, and wash the plate 5 times.
5. **Enzyme Addition:** Add 50 μL of enzyme conjugate to each well except blank.
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 to each well.
9. **Reading:** Measure OD at 450 nm within 15 minutes of stopping the reaction.
**Notes:**
- Allow the kit to equilibrate at room temperature for 15–30 minutes before use.
- If the enzyme reagent is unsealed, store it in a sealed bag.
- Wash solution may crystallize; heat gently if needed.
- Use a pipette accurately and avoid cross-contamination.
- Always prepare a standard curve and run duplicates.
- Avoid exposure of TMB to light.
- Follow the manual strictly. Results must be based on microplate reader readings.
- Treat all samples and waste as biohazardous materials.
- Do not mix reagents from different batches.
- In case of conflict, the English manual takes precedence.
**Kit Performance:**
- Linear correlation coefficient (R) ≥ 0.95
- Intra-batch variation < 9%, inter-batch variation < 11%
- Detection range: 0.2 IU/L – 6 IU/L
This detailed guide ensures accurate and reliable detection of CgA in various biological samples. Proper handling and adherence to the protocol are essential for optimal results.
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