**Human Glycosylated Hemoglobin A1c (GHbA1c) ELISA Kit Instructions**
*Guangrui Bio – Domestic High-Quality ELISA Kit Supplier*
This reagent is for **research purposes only**. The kit is designed to quantitatively determine the concentration of **glycosylated hemoglobin A1c (GHbA1c)** in human serum, plasma, and other related biological fluids.
### **Principle of the Assay**
The GHbA1c ELISA kit employs a **double-antibody sandwich immunoassay** method. The microtiter plate is pre-coated with a specific antibody against GHbA1c. After incubation with the sample, the GHbA1c in the sample binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming a complex of antibody-antigen-enzyme-labeled antibody. Following a wash step, TMB substrate is added, which turns blue under the catalytic action of HRP. The reaction is stopped by adding an acidic solution, causing the color to shift to yellow. The intensity of the color is directly proportional to the GHbA1c concentration in the sample. The absorbance is measured at 450 nm using a microplate reader, and the GHbA1c concentration is determined from a standard curve.
### **Kit Components**
- **48-well configuration**: 1×48 enzyme-labeled plate, 1 sealing film, 2 standard solutions (0.5 ml × 1 bottle), 2 standard dilutions (1.5 ml × 1 bottle), 2 enzyme reagents (3 ml × 1 bottle), 2 developer solutions (A & B, 3 ml × 1 bottle), 1 washing solution (20 ml × 20 times), 1 sample dilution (3 ml × 1 bottle).
- **96-well configuration**: 1×96 enzyme-labeled plate, 2 sealing films, 2 standard solutions (0.5 ml × 1 bottle), 2 standard dilutions (1.5 ml × 1 bottle), 2 enzyme reagents (6 ml × 1 bottle), 2 developer solutions (A & B, 6 ml × 1 bottle), 1 washing solution (20 ml × 30 times), 1 sample dilution (6 ml × 1 bottle).
All components should be stored at **2–8°C**.
### **Sample Preparation**
- **Serum**: Collect blood at room temperature, allow to clot for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. Discard any precipitate after storage.
- **Plasma**: Use anticoagulants such as EDTA, sodium citrate, or heparin. Mix well, centrifuge, and collect supernatant.
- **Urine**: Collect using sterile tubes, centrifuge, and discard any precipitate.
- **Cell culture supernatant**: Centrifuge to remove debris; for intracellular components, lyse cells via repeated freeze-thaw cycles before centrifuging.
- **Tissue samples**: Homogenize in PBS, centrifuge, and collect supernatant. Store at 2–8°C if not used immediately.
### **Procedure**
1. **Standard Dilution**: Prepare a 5-fold serial dilution of the standard solution in the designated wells.
2. **Sample Addition**: Add 40 μl of sample diluent and 10 μl of sample to each test well.
3. **Incubation**: Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing**: Wash 5 times with diluted washing solution.
5. **Enzyme Addition**: Add 50 μl of HRP-labeled antibody to each well except blank.
6. **Second Incubation**: Repeat incubation at 37°C for 30 minutes.
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. **Measurement**: Read OD values at 450 nm within 15 minutes.
### **Notes and Precautions**
- Allow the kit to equilibrate at room temperature for 15–30 minutes before use.
- Avoid cross-contamination; use a new sealing film for each experiment.
- Ensure accurate pipetting; use a multichannel pipette for large numbers of samples.
- Always run standards in duplicate and prepare a standard curve. If the sample OD exceeds the highest standard, dilute the sample and retest.
- Keep all reagents away from light.
- Dispose of all waste materials as biohazardous.
### **Calculation**
Plot the standard curve using GHbA1c concentrations vs. OD values. Calculate the sample concentration using linear regression or interpolation. Multiply by the dilution factor to obtain the actual value.
### **Performance**
- **Sensitivity**: 100 nmol/L – 2400 nmol/L
- **Intra-batch CV < 9%**, **Inter-batch CV < 11%**
- **Correlation coefficient (R²) ≥ 0.95**
### **Storage and Shelf Life**
- **Storage Conditions**: 2–8°C
- **Shelf Life**: 6 months from the date of manufacture.
This kit provides a reliable and sensitive method for measuring GHbA1c levels in clinical research settings. Always follow the manufacturer’s instructions carefully for accurate results.
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