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Human hs-CRP ELISA Kit
**Human hs-CRP ELISA Kit – For the quantitative in vitro determination of Human high sensitivity C-Reactive Protein concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
This kit is designed for research purposes only and is not intended for diagnostic or therapeutic use. The assay is based on the enzyme-linked immunosorbent assay (ELISA) principle, which allows for the accurate quantification of human hs-CRP levels. The reaction involves a colorimetric detection system where the addition of a stop solution changes the color from blue to yellow. The optical density (OD) at 450 nm is then measured using a spectrophotometer.
To determine the concentration of hs-CRP in the sample, a set of calibration standards is included in the kit. These standards are used to generate a standard curve by plotting OD values against known concentrations. The sample concentrations are calculated by comparing their OD values 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 and assay immediately or store at -20°C. Thaw only once.
*Note: Ensure proper centrifugation and avoid hemolysis or granules in the samples.*
**Materials Required but Not Supplied:**
1. Incubator at 37°C
2. Microplate reader capable of measuring absorbance at 450 nm
3. Precision pipettes, disposable tips, and absorbent paper
4. Distilled or deionized water
**Reagents Provided:**
- 96 determinations / 48 determinations
- Microtiter Strip Plate (12 x 8 strips / 12 x 4 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 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: 400, 200, 100, 50, 25, 12.5 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 kit lots. Standards, conjugate, and plates are matched for optimal performance. Use only manufacturer-supplied reagents.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use. Avoid using water baths for thawing.
3. Do not use reagents past their expiration date.
4. Store unused strips in sealed bags with desiccant at 2–8°C.
5. Wear gloves during the procedure. Handle all samples carefully.
6. Inactivate waste for 30 minutes before disposal.
7. Substrate solutions should be clear; discard if discolored.
8. Substrate B contains 20% acetone—keep away from heat and flame.
**Reagent Preparation:**
- Wash Solution (1X): Dilute 1 volume of 20X Wash Solution with 19 volumes of distilled or deionized water. Stable for 1 month at 2–8°C.
**Assay Procedure:**
1. Prepare all reagents before starting. Run standards and samples in duplicate.
2. Add 50 µl of standard or sample to each well. 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 microtiter plate 4 times (manual or automated).
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. Measure OD at 450 nm. Subtract blank OD from all values. Plot standard curve and calculate sample concentrations.
**Performance Characteristics:**
- Intra-assay and Inter-assay CV < 15%
- Assay range: 12.5 – 400 ng/mL
- Sensitivity: <10 ng/mL
- Cross-reactivity: No significant cross-reaction with other proteins
- Storage: 2–8°C (frequent use); 6 months at -20°C
**Important Notes:**
- Each user should generate their own standard curve due to possible variations in technique or environmental factors.
- Always follow safety protocols when handling biological samples.
- Proper storage and handling of reagents ensure reliable results.
For detailed instructions, refer to the complete user manual provided.