**Human Glycosylated Hemoglobin A1c (GHbA1c) ELISA Kit Instructions**
*Guangrui Bio – Domestic High-Quality ELISA Kit Supplier*
This reagent is intended for **research purposes only**. The kit is designed to quantitatively determine the level of **glycosylated hemoglobin A1c (GHbA1c)** in human serum, plasma, and related liquid samples.
**Experimental Principle**
The kit employs a **double-antibody sandwich ELISA** method. A microplate is pre-coated with a specific antibody against GHbA1c. After adding the sample, the target antigen binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming an immune complex. Following washing steps, TMB substrate is introduced, leading to a color change that correlates with the GHbA1c concentration. The reaction is stopped with a stop solution, and absorbance is measured at 450 nm using a microplate reader. The GHbA1c concentration is calculated from a standard curve.
**Kit Composition**
- 48-well / 96-well configuration
- Coated microplates
- Standard solutions (2700 nmol/L)
- Enzyme-labeled reagents
- Sample diluent
- TMB substrate A & B
- Washing buffer (concentrated, 20x)
- Sealing films and bags
- Storage: 2–8°C
**Sample Preparation**
- **Serum**: Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma**: Use anticoagulants like EDTA or heparin, mix, and centrifuge similarly.
- **Urine**: Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell culture supernatant**: Centrifuge to remove debris. For intracellular components, lyse cells via freeze-thaw cycles.
- **Tissue**: Homogenize in PBS, centrifuge, and collect supernatant.
**Operation Steps**
1. **Standard Dilution**: Prepare serial dilutions of the standard on the plate.
2. **Sample Addition**: Add 40 μL of sample diluent and 10 μL of sample (final 5× dilution).
3. **Incubation**: Seal and incubate at 37°C for 30 minutes.
4. **Washing**: Wash 5 times with diluted washing buffer.
5. **Enzyme Addition**: Add 50 μL of HRP-labeled antibody.
6. **Second Incubation**: Repeat incubation at 37°C for 30 minutes.
7. **Color Development**: Add 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 at 450 nm within 15 minutes.
**Notes**
- Allow the kit to reach room temperature before use.
- Avoid cross-contamination; use a new sealing film for each experiment.
- Ensure accurate pipetting and proper calibration.
- Always prepare a standard curve and perform duplicates.
- Store all waste as biohazardous material.
- Do not mix reagents from different batches.
**Calculation**
Plot the standard curve using GHbA1c concentrations vs. OD values. Calculate sample concentration by interpolating from the curve or using linear regression. Multiply by the dilution factor for final results.
**Performance**
- Correlation coefficient (R) ≥ 0.95
- Intra-batch CV < 9%, Inter-batch CV < 11%
- Detection range: 100–2400 nmol/L
**Storage & Expiration**
- Store at 2–8°C
- Shelf life: 6 months from date of manufacture
This ELISA kit provides a reliable and sensitive method for measuring GHbA1c levels, supporting clinical and research applications. Always follow the instructions carefully for accurate and reproducible results.
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