**Human Aspartate Aminotransferase (GOT/GPT) ELISA Kit – Instructions for Use**
**Kit Specifications:**
This kit is available in 48-well or 96-well configurations. It includes:
- Standard Diluent: 1.5 mL × 1 vial
- Enzyme Standard Reagent: 3 mL × 1 vial (for 48 wells) / 6 mL × 1 vial (for 96 wells)
- Microplate Sealing Film: 2 pieces (for 48 wells) / 2 pieces (for 96 wells)
- Standard: 0.5 mL × 1 vial, 2700 ng/L
- Enzyme Standard: 1×48 / 1×96
- Sample Diluent: 3 mL × 1 vial (for 48 wells) / 6 mL × 1 vial (for 96 wells)
- Developer A: 3 mL × 1 vial (for 48 wells) / 6 mL × 1 vial (for 96 wells)
- Chromogen B: 3 mL × 1 vial (for 48 wells) / 6 mL × 1 vial (for 96 wells)
- Stop Solution: 3 mL × 1 vial (for 48 wells) / 6 mL × 1 vial (for 96 wells)
- Concentrated Washing Solution: 20 mL × 20 times (for 48 wells) / 20 mL × 30 times (for 96 wells)
**Storage Conditions & Shelf Life:**
- Store the kit at 2–8°C.
- Shelf life: 6 months from the date of manufacture.
**Purpose:**
This ELISA kit is designed to quantitatively determine the concentration of human Aspartate Aminotransferase (GOT/GPT) in serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids.
**Principle of Operation:**
The kit utilizes a sandwich ELISA method. The microtiter plate is pre-coated with a specific antibody against human GOT/GPT. After incubation with the sample, the antigen binds to the immobilized antibody. A HRP-conjugated secondary antibody is then added, forming a complex. TMB substrate is used to develop the color, which is stopped by an acidic solution. The intensity of the color is directly proportional to the concentration of GOT/GPT in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, and the concentration is calculated based on a standard curve.
**Sample Preparation:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix well, centrifuge, and collect the supernatant.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
- **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via repeated freezing and thawing before centrifugation.
- **Tissue Homogenate:** Weigh the tissue, add PBS, homogenize, centrifuge, and collect the supernatant.
**Operation Steps:**
1. Prepare standard dilutions in a serial manner (1800 ng/L, 1200 ng/L, 600 ng/L, 300 ng/L, 150 ng/L).
2. Add 40 µL of sample diluent and 10 µL of sample to each test well (final dilution 5x).
3. Seal the plate and incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted washing buffer.
5. Add 50 µL of enzyme-labeled reagent to each well except blank.
6. Incubate again at 37°C for 30 minutes.
7. Wash the plate again.
8. Add 50 µL of TMB A and TMB B, incubate at 37°C for 15 minutes.
9. Stop the reaction by adding 50 µL of stop solution.
10. Measure OD at 450 nm within 15 minutes.
**Notes:**
- Let the kit equilibrate to room temperature before use.
- Avoid cross-contamination; use a new sealing film for each experiment.
- Do not mix reagents from different batches.
- Ensure accurate pipetting and avoid light exposure during color development.
- All waste should be treated as biohazardous material.
**Performance:**
- Linear range: 0.2 IU/L – 6 IU/L
- Correlation coefficient (R²): ≥ 0.95
- Intra-assay CV < 9%, Inter-assay CV < 11%
**Technical Support:**
We offer free technical support during working hours. Please contact us for assistance with sample testing and experimental design.
**Note:** This kit is for research use only.
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