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**Human TSP-1 ELISA Kit – For the Quantitative In Vitro Determination of Human Thrombospondin 1 Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Biological Fluids**
**FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
This package insert must be read carefully before using the ELISA kit. The kit is intended for research purposes only and not for diagnostic or therapeutic procedures. 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 TSP-1 in the sample, this ELISA Kit includes a set of calibration standards. These standards are run alongside the samples and allow the operator to generate a standard curve of Optical Density (OD) versus TSP-1 concentration. The TSP-1 concentration in the samples is then determined by comparing their OD values to the standard curve.
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**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 for 20 minutes at approximately 2000×g. Remove serum and assay immediately or aliquot and store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma**: Collect plasma using heparin as an anticoagulant. Centrifuge within 30 minutes of collection at 2000×g for 30 minutes at 2–8°C. Store at -20°C. Avoid repeated freeze-thaw cycles.
- **Cell culture supernatants, tissue homogenates, and other biological fluids**: Remove particulates by centrifugation. Assay immediately or aliquot and store at -20°C. Ensure adequate centrifugation and avoid hemolysis or granules.
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**MATERIALS REQUIRED BUT NOT SUPPLIED**
1. 37°C incubator
2. Standard microplate reader capable of measuring absorbance at 450 nm
3. Precision pipettes, disposable pipette tips, and absorbent paper
4. Distilled or deionized water
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**REAGENTS PROVIDED (Stored at 2–8°C)**
| Name | 96 Determinations | 48 Determinations |
|------|-------------------|-------------------|
| MicroELISA Stripplate | 12*8 strips | 12*4 strips |
| Standard (6 vials) | 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 |
**Note:**
- Standard concentrations: 2000, 1000, 500, 250, 125, 62.5 ng/mL
- If sample values exceed the highest standard, dilute with Sample Diluent and repeat the test.
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**PRECAUTIONS AND GUIDELINES**
1. All reagents should reach room temperature (20–25°C) before use. Do not use water baths to thaw samples or reagents.
2. Do not use any kit components past their expiration date.
3. Only use deionized or distilled water for dilutions.
4. Keep microtiter plates in their sealed bags until needed. Unused strips should be stored at 2–8°C with desiccant.
5. Use fresh disposable pipette tips for each transfer to avoid cross-contamination.
6. Disposable items must be treated as potentially hazardous. Handle all blood-derived products with caution and follow good lab practices.
7. Dispose of all samples according to local regulations. Allow liquid to stand for at least 30 minutes to inactivate viruses before disposal.
8. Substrate Solution A and B may be easily contaminated. Substrate B contains 20% acetone—keep away from heat or flame.
9. Allow all reagents to reach room temperature before starting the assay.
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**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 1 month.
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**ASSAY PROCEDURE**
1. Prepare all reagents before starting the assay.
2. Add 50 µL of Standard or Sample to appropriate wells (excluding the blank well). Cover with an adhesive strip and incubate for 60 minutes at 37°C.
3. Wash the microtiter plate 4 times. Manual washing involves aspirating and filling with Wash Solution (1X), repeating four times. After final wash, invert and blot dry.
4. Add 50 µL of Chromogen Solution A and 50 µL of Chromogen Solution B to each well. Incubate for 15 minutes at 37°C, protecting from light.
5. Add 50 µL of Stop Solution to each well. Read OD at 450 nm using a microplate reader.
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**DATA ANALYSIS**
1. Generate a standard curve by plotting average OD values (450 nm) of the six standards against their concentrations.
2. Calculate the mean OD value for each standard and sample. Subtract the blank OD value before interpretation.
3. Use graph paper or software to construct the standard curve. Locate the OD value on the Y-axis and draw a horizontal line to intersect the curve. From there, draw a vertical line to the X-axis to determine the TSP-1 concentration.
4. Variations in technique, incubation time, or kit age can affect results. Each user should create their own standard curve.
5. Intra-assay and inter-assay CV (%) are less than 15%.
6. Assay range: 62.5 ng/mL – 2000 ng/mL.
7. Sensitivity: Minimum detectable dose < 10 ng/mL.
8. Cross-reactivity: No significant cross-reactivity observed.
9. Storage: 2–8°C (for frequent use); 6 months at -20°C.
**Please read all instructions carefully before performing the assay.**
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