**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. This kit is intended for laboratory research use only and is not suitable for diagnostic or therapeutic purposes.**
The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm using a spectrophotometer. To determine the concentration of GSTt1 in the sample, the kit includes a set of calibration standards. These standards are run alongside the samples to generate a standard curve, plotting optical density (OD) against known GSTt1 concentrations. The sample concentration is then calculated by comparing its OD value to this curve.
**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 at 2000 ×g for 20 minutes. 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 2000 ×g, 2–8°C. Avoid freeze-thaw cycles.
- **Cell culture supernatants, tissue homogenates, and other biological fluids:** Centrifuge to remove particulates, then assay immediately or store at -20°C. Ensure no hemolysis or granules are present.
**Materials Required (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 (Store at 2–8°C):**
- 96-well microplate (12×8 strips)
- 6 standard vials (0.5 ml/vial)
- Sample diluent (6.0 ml)
- HRP-Conjugate reagent (10.0 ml)
- 20X Wash solution (25 ml)
- Chromogen A (6.0 ml), Chromogen B (6.0 ml)
- Stop solution (6.0 ml)
- Closure plate membrane (2 pieces)
- User manual and sealed bags
**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:**
- Do not mix reagents from different kits.
- Allow all reagents to reach room temperature before use.
- Do not use water baths to thaw samples or reagents.
- Do not use reagents beyond their expiration date.
- Only use deionized or distilled water.
- Keep unused strips in the storage bag with desiccant.
- Use fresh pipette tips for each transfer.
- Handle all blood-derived samples as potentially infectious.
- Wear gloves during the procedure.
- Dispose of all samples properly after inactivation (e.g., 30 minutes in disinfectant).
**Reagent Preparation:**
- Wash solution (1X): Dilute 1 volume of 20X wash solution with 19 volumes of water. Store at 2–8°C for 1 month.
**Assay Procedure:**
1. Prepare all reagents before starting.
2. Add 50 µL of standard or sample to appropriate wells. Blank well receives nothing.
3. Add 100 µL HRP-conjugate reagent to all wells except blank. Incubate for 60 minutes at 37°C.
4. Wash 4 times manually or automatically.
5. Add 50 µL chromogen A and 50 µL chromogen B to each well. Incubate for 15 minutes at 37°C, away from light.
6. Add 50 µL stop solution. Color should turn yellow. If green or uneven, gently mix.
7. Read OD at 450 nm. Generate standard curve by plotting average OD vs. concentration.
**Interpretation:**
- Calculate mean OD for each standard and sample.
- Subtract blank OD from all readings.
- Plot standard curve and interpolate sample values.
- Intra- and inter-assay CV <15%.
- Assay range: 37.5–1200 pg/mL.
- Sensitivity: <1.0 pg/mL.
- No cross-reactivity observed.
**Storage:**
- 2–8°C for frequent use; up to 6 months at -20°C.
**Note:** Always follow proper lab safety protocols and dispose of waste according to local regulations. This kit is not for diagnostic use.
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