**Human Th17 ELISA Kit – For the Quantitative In Vitro Determination of Human Th17 Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Biological Fluids**
**For Laboratory Research Use Only. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.**
Before using this product, please read the entire package insert carefully. This kit is designed for research purposes only and should not be used for diagnostic or clinical testing. The enzyme-linked immunosorbent assay (ELISA) method enables the accurate quantification of human Th17 levels in various biological samples.
### **Intended Use and Test Principle**
This Human Th17 ELISA Kit is intended for laboratory research use only and is not suitable for diagnostic or therapeutic applications. The assay is based on a sandwich ELISA format, where the captured Th17 molecules are detected using a specific antibody conjugated with horseradish peroxidase (HRP). A chromogenic substrate is added, leading to a color change that is measured spectrophotometrically at 450 nm. By comparing the optical density (OD) of the samples to a standard curve, the concentration of Th17 in each sample can be accurately determined.
### **Sample Collection and Storage**
- **Serum**: Collect using a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C. Centrifuge at 2000×g for 20 minutes. Assay 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. Avoid repeated freezing.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or granules are present.
### **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**
All reagents are stored at 2–8°C. Refer to the expiration date on the label.
| Name | 96 Determinations | 48 Determinations |
|------|-------------------|-------------------|
| Microtiter Strip Plate | 12×8 strips | 12×4 strips |
| Standard (6 vials, 0.5 mL/vial) | 0.5 mL/vial | 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 Solution A | 6.0 mL | 3.0 mL |
| Chromogen Solution B | 6.0 mL | 3.0 mL |
| Stop Solution | 6.0 mL | 3.0 mL |
| Closure Plate Membrane | 2 | 2 |
| User Manual | 1 | 1 |
| Sealed Bags | 1 | 1 |
*Standard concentrations: 50, 25, 12.5, 6.25, 3.12, 1.56 ng/mL.*
*If sample values exceed the highest standard, dilute with sample diluent and repeat the assay.*
### **Precautions**
1. Do not mix reagents from different kits. Each component is calibrated for optimal performance.
2. Allow all reagents to reach room temperature (20–25°C) before use. Do not thaw using water baths.
3. Do not use reagents past their expiration date.
4. Only use deionized or distilled water for dilution.
5. Keep unused microtiter plate strips in sealed bags with desiccant.
6. Use fresh pipette tips for each transfer to prevent cross-contamination.
7. Avoid using disposable knives derived from rat blood due to potential infectious risks.
8. Liquid waste must be treated with 1% sodium hypochlorite for at least 30 minutes before disposal.
9. Substrate solution may be contaminated; discard if it appears bluish.
10. Chromogen B contains 20% acetone—keep away from heat and flame.
11. Bring all reagents to room temperature before starting the assay.
### **Reagent Preparation and Storage**
**Wash Solution (1X)**: Mix 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 beginning.
2. Add 100 µL of standards, controls, and samples to appropriate wells.
3. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the plate 4 times using either manual or automated washing methods.
- *Manual*: Fill each well with 350 µL of 1X Wash Solution, aspirate, and repeat four times.
- *Automated*: Aspirate and wash four times, adjusting brush settings for maximum liquid removal.
5. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protecting 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. Measure OD at 450 nm within 15 minutes using a microplate reader.
### **Calculation of Results**
- Plot average OD values of standards vs. their concentrations on a graph.
- Subtract blank OD from sample OD values before interpretation.
- Locate the OD value on the Y-axis and draw a horizontal line to intersect the standard curve. Read the corresponding concentration on the X-axis.
- Each user should generate their own standard curve.
- Intra-assay CV < 15%, Inter-assay CV < 15%.
- Assay range: 1.56 – 50 ng/mL.
- Sensitivity: < 1.0 ng/mL.
- Cross-reactivity: No significant cross-reactivity with other cytokines.
- Storage: 2–8°C (frequent use); -20°C (long-term storage, up to 6 months).
**Always follow safety protocols and dispose of waste properly.**
**For research use only.**
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