**Human GSTt1 ELISA Kit – For the quantitative in vitro determination of Human Glutathione S-Transferase theta 1 concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and OTHER body fluids. FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.**
This ELISA kit is specifically designed for research purposes and must not be used for diagnostic or clinical applications. The kit allows for the accurate measurement of GSTt1 levels by detecting color changes that occur during the reaction. The Stop Solution turns the color from blue to yellow, and the intensity is measured at 450 nm using a spectrophotometer.
To determine the concentration of GSTt1 in your samples, the kit includes a set of calibration standards. These standards are run alongside the samples to generate a standard curve, which is essential for calculating the exact concentration of GSTt1 in each sample. This process ensures reliable and reproducible results.
**Sample Collection and Storage:**
- **Serum:** Use a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation (20 minutes at ~2000×g). Remove serum and assay immediately, or aliquot and store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma:** Collect using heparin as an anticoagulant. Centrifuge within 30 minutes at 2–8°C for 30 minutes at 2000×g. Avoid freezing and thawing multiple times.
- **Cell culture supernatants, tissue homogenates, and other biological fluids:** Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or debris is present in the samples.
**Materials Required but Not Supplied:**
- 37°C incubator
- Microplate reader capable of measuring absorbance at 450 nm
- Precision pipettes, disposable tips, and absorbent paper
- Distilled or deionized water
**Reagents Provided (Stored at 2–8°C):**
- 96-well microplate (12×8 strips)
- Standard (6 vials, 0.5 ml/vial)
- Sample diluent (6.0 ml / 3.0 ml)
- HRP-Conjugate reagent (10.0 ml / 5.0 ml)
- 20X Wash solution (25 ml / 15 ml)
- Chromogen A (6.0 ml / 3.0 ml)
- Chromogen B (6.0 ml / 3.0 ml)
- Stop solution (6.0 ml / 3.0 ml)
- Closure plate membrane (2 units)
- User manual (1 copy)
- Sealed bags (1 unit)
**Standard Concentrations:** 1200, 600, 300, 150, 75, 37.5 pg/mL. If sample values exceed the highest standard, dilute with sample diluent and repeat the assay.
**Precautions:**
- Never substitute reagents between different kit lots. All components are calibrated for optimal performance.
- Allow all reagents to reach room temperature (20–25°C) before use.
- Do not use expired reagents.
- Use only deionized or distilled water.
- Store unused strips in their sealed pouch with desiccant.
- Wear gloves when handling plasma or any potentially infectious material.
- Dispose of all waste properly after inactivation for at least 30 minutes.
- Handle substrate solutions carefully—avoid contamination and exposure to heat or flame.
**Reagent Preparation:**
- Wash Solution (1X): Dilute 1 volume of 20X wash solution with 19 volumes of deionized or distilled water. Store at 2–8°C for up to one month.
**Assay Procedure:**
1. Prepare all reagents before starting the assay. Run standards and samples in duplicate.
2. Add 50 µL of standard or sample to appropriate wells. Blank well receives no addition.
3. Add 100 µL of HRP-conjugate reagent to all wells except blank. Cover with adhesive strip 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. Color changes from blue to yellow. If uneven, gently tap the plate.
7. Read OD at 450 nm. Generate a standard curve by plotting average OD vs. concentration.
**Interpretation:**
- Calculate mean OD for each standard and sample.
- Subtract blank OD from all values.
- Determine sample concentration by comparing its OD to the standard curve.
- Intra-assay and inter-assay CVs are <15%.
- Assay range: 37.5–1200 pg/mL.
- Sensitivity: <1.0 pg/mL.
- No cross-reactivity or interference observed.
- Store at 2–8°C (frequent use) or -20°C (long-term storage).
**Note:** Always read the full user manual before beginning the assay. Follow all safety protocols and handle all materials with care.
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