**Human GSTM1 ELISA Kit – For the Quantitative In Vitro Determination of Human Glutathione S-Transferase Mu 1 in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and OTHER Biological Fluids**
*For Laboratory Research Use Only. Not Intended for Diagnostic or Therapeutic Procedures.*
This kit is designed to quantitatively measure the levels of Human Glutathione S-Transferase mu 1 (GSTM1) in various biological samples using an Enzyme-Linked Immunosorbent Assay (ELISA). The assay is based on a competitive binding principle where GSTM1 in the sample competes with a GSTM1-HRP conjugate for binding sites on the immobilized antibodies. The reaction is terminated by adding a stop solution, and the color intensity is measured at 450 nm. A standard curve is generated using known concentrations of GSTM1, allowing for accurate quantification of unknown samples.
**Intended Use:**
This Human GSTM1 ELISA Kit is intended for laboratory research purposes only and is not suitable for use in diagnostic or therapeutic procedures.
**Test Principle:**
The kit uses a sandwich ELISA format. The microtiter plate is pre-coated with specific antibodies against GSTM1. After incubation with standards and samples, a horseradish peroxidase (HRP)-labeled antibody is added. Following washing steps, a chromogenic substrate is introduced, and the color development is stopped. The optical density (OD) is measured at 450 nm, and the concentration of GSTM1 is determined by comparing the OD values to the standard curve.
**Sample Collection and Storage:**
- **Serum:** Collect using a serum separator tube. Allow clotting for 2 hours at room temperature or overnight at 4°C before centrifuging at 2000×g for 20 minutes. Remove serum and analyze immediately or store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma:** Collect using heparin as an anticoagulant. Centrifuge within 30 minutes at 2000×g, 2–8°C. Store at -20°C if not used immediately.
- **Cell Culture Supernatant, Tissue Homogenate, and Other Biological Fluids:** Centrifuge to remove particulates. Analyze immediately or store at -20°C. Avoid repeated freezing and thawing.
*Note: Ensure proper centrifugation and avoid hemolysis or granulation in samples.*
**Materials Required but Not Supplied:**
1. Incubator set at 37°C
2. Microplate reader capable of measuring absorbance at 450 nm
3. Precision pipettes, disposable tips, and absorbent paper
4. Distilled or deionized water
**Reagents Provided (Stored at 2–8°C):**
- Microtiter Strip Plate (12×8 or 12×4 strips)
- Standard (6 vials, 0.5 ml/vial) – 1200, 600, 300, 150, 75, 37.5 pg/mL
- Sample Diluent (6.0 ml or 3.0 ml)
- HRP-Conjugate Reagent (10.0 ml or 5.0 ml)
- 20X Wash Solution (25 ml or 15 ml)
- Chromogen Solution A (6.0 ml or 3.0 ml)
- Chromogen Solution B (6.0 ml or 3.0 ml)
- Stop Solution (6.0 ml or 3.0 ml)
- Closure Plate Membrane (2 units)
- User Manual (1 copy)
- Sealed Bags (1 unit)
**Important Notes:**
- Do not mix reagents from different kits or lots.
- Allow all reagents to reach room temperature (20–25°C) before use.
- Do not use reagents beyond their expiration date.
- Always use deionized or distilled water for dilutions.
- Keep unused strip wells in sealed bags with desiccants at 2–8°C.
- Use fresh pipette tips for each transfer to prevent contamination.
- Handle plasma as potentially hazardous material. Wear gloves during the procedure.
- Waste should be inactivated for at least 30 minutes before disposal.
- Substrate solutions are sensitive; discard if discolored.
- Chromogen B contains 20% acetone—keep away from heat and flame.
**Assay Procedure:**
1. Prepare all reagents before starting. Add standards and samples in duplicate.
2. Add 50 µL of standard or sample to each well. Blank well receives no addition.
3. Add 100 µL of HRP-conjugate to all wells except blank. Cover and incubate for 60 minutes at 37°C.
4. Wash the plate 4 times manually or automatically.
5. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protected from light.
6. Add 50 µL of Stop Solution to each well. The color changes from blue to yellow. Gently mix if necessary.
7. Measure OD at 450 nm. Generate a standard curve using average OD values of standards. Calculate sample concentrations accordingly.
**Performance Characteristics:**
- Sensitivity: <1.0 pg/mL
- Range: 37.5–1200 pg/mL
- Intra-assay CV <15%, Inter-assay CV <15%
- No significant cross-reactivity with other proteins
**Storage:**
- Short-term: 2–8°C (for frequent use)
- Long-term: -20°C (up to 6 months)
**Safety and Disposal:**
Handle all reagents and samples with care. Follow local regulations for waste disposal. Ensure proper personal protective equipment (PPE) is worn throughout the procedure.
**Limitations:**
This kit is not approved for clinical diagnostics. Results should not be used for medical decision-making. Each user should generate their own standard curve for optimal accuracy.
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