**Human Chromoprotein A (CgA) ELISA Kit Instruction Manual**
**Kit Specification:**
This ELISA kit is available in 48-well or 96-well configurations. The standard reagent is provided as 1.5 mL × 1 bottle. The enzyme standard reagent comes in 3 mL × 1 bottle (for 48-well) or 6 mL × 1 bottle (for 96-well). This reagent is for research use only.
**Standard Curve Preparation:**
To determine the concentration of CgA in the sample, a standard curve is plotted by plotting the standard concentrations on the x-axis and the corresponding OD values on the y-axis. Alternatively, a linear regression equation can be calculated using the standard concentrations and their respective OD values. The OD value of the sample is then substituted into the equation to calculate its concentration, which is multiplied by the dilution factor to obtain the actual sample concentration.
**Kit Components:**
- 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 buffer: 3 mL × 1 bottle (48) / 6 mL × 1 bottle (96)
- Concentrated wash solution: (20 mL × 20 times) × 1 bottle (48) / (20 mL × 30 times) × 1 bottle (96)
**Storage Conditions & Expiration:**
- Storage: 2–8°C
- Shelf Life: 6 months from the date of manufacture
**Principle of the Assay:**
The Human Chromogranin A (CgA) ELISA Kit utilizes the sandwich immunoassay method. The microtiter plate is pre-coated with a specific antibody against CgA. After adding the sample and the HRP-conjugated secondary antibody, a complex is formed. After washing, TMB substrate is added, and the color develops in proportion to the amount of CgA present. The reaction is stopped, and the absorbance at 450 nm is measured. The concentration of CgA in the sample is determined based on a standard curve.
**Purpose:**
This kit is designed to quantify the levels of Chromogranin A (CgA) in human serum, plasma, urine, cell culture supernatants, and other biological fluids.
**Sample Preparation Guidelines:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix well and centrifuge as above.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells by repeated freezing and thawing.
- **Tissue Samples:** Homogenize in PBS, centrifuge, and collect the supernatant. Store at 2–8°C or freeze at -20°C if not used immediately.
- **Avoid repeated freeze-thaw cycles. Do not use samples containing NaN3.**
**Operating Procedure:**
1. **Standard Dilution:** Prepare serial dilutions of the standard.
2. **Loading:** Add 40 µL of sample diluent and 10 µL of sample to each well (final dilution 5x).
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Wash the plate 5 times with diluted wash buffer.
5. **Enzyme Addition:** Add 50 µL of enzyme-labeled reagent to each well (except blank wells).
6. **Second Incubation:** Incubate again at 37°C for 30 minutes.
7. **Color Development:** Add 50 µL of TMB A and B, incubate for 15 minutes at 37°C.
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.
- If the concentrated wash solution crystallizes, warm it gently in a water bath.
- Use a pipette for accuracy; avoid cross-contamination.
- Always run a standard curve in duplicate. If the sample OD exceeds the highest standard, dilute the sample before testing.
- Keep all components sealed and away from light.
- Follow instructions strictly. Results should be based on the microplate reader readings.
- Treat all waste materials as biohazardous.
- Do not mix reagents from different batches.
- In case of conflict between languages, the English manual takes precedence.
**Kit Performance:**
- Correlation coefficient (R) of the standard curve ≥ 0.95
- Intra-batch CV < 9%, Inter-batch CV < 11%
- Detection range: 0.2 IU/L – 6 IU/L
This guide ensures accurate and reliable detection of Human Chromogranin A (CgA) in various biological samples. Proper handling and following the protocol are essential for optimal results.
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