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Human ICAM-3 ELISA Kit
**Human ICAM-3 ELISA Kit – For the quantitative in vitro determination of Human Intercellular Adhesion Molecule 3 (ICAM-3) concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. This kit is intended for laboratory research use only and not for diagnostic or therapeutic purposes.**
This ELISA kit is designed to measure the concentration of ICAM-3 using a colorimetric detection method. The reaction involves binding of ICAM-3 to specific antibodies coated on the microtiter plate, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody. After washing, a chromogenic substrate is added, resulting in a blue color change. The reaction is stopped by adding a stop solution, which turns the color yellow. The optical density (OD) at 450 nm is then measured using a spectrophotometer.
To ensure accurate results, a set of calibration standards is included in the kit. These standards are run alongside the samples, allowing the user to generate a standard curve. By comparing the OD values of the unknown samples to this curve, the concentration of ICAM-3 can be precisely determined.
**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 centrifuging at 2000×g for 20 minutes. Remove the 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 freezing and thawing.
- **Cell culture supernatants, tissue homogenates, and other biological fluids:** Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or contamination occurs during collection.
**Materials Required (Not Included):**
- 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):**
- MicroELISA strip plate (12 x 8 strips / 12 x 4 strips)
- Standards (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 units)
- User Manual (1 copy)
- Sealed Bags (1 unit)
**Precautions:**
- Do not mix reagents from different kits. All components are calibrated for optimal performance.
- Allow all reagents to reach room temperature (20–25°C) before use. Avoid using water baths for thawing.
- Do not use reagents beyond their expiration date.
- Always use deionized or distilled water for dilutions.
- Store unused strips in sealed bags with desiccant at 2–8°C.
- Use fresh pipette tips for each transfer to prevent cross-contamination.
- Wear appropriate personal protective equipment (PPE), including gloves and lab coats, when handling biological samples.
**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 one month.
**Assay Procedure:**
1. Prepare all reagents before starting. Run standards and samples in duplicate.
2. Add 50 μL of standard or sample to the designated wells. Blank well receives no addition.
3. Add 100 μL of HRP-Conjugate Reagent to all wells except the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the plate 4 times manually or automatically. Ensure complete removal of unbound conjugate.
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. The color should turn yellow. If uneven, gently tap the plate.
7. Measure OD at 450 nm. Plot the average OD of each standard against its concentration to create a standard curve.
8. Calculate the mean OD for each sample and subtract the blank value before determining ICAM-3 concentration.
**Performance Characteristics:**
- Sensitivity: <10 ng/mL
- Detection Range: 30–960 ng/mL
- Intra-assay CV: <15%
- Inter-assay CV: <15%
- Cross-reactivity: No significant cross-reactivity with other molecules
**Storage:**
- Store at 2–8°C for frequent use; for long-term storage, keep at -20°C for up to six months.
**Disposal:**
- Follow local regulations for biohazardous waste. Ensure proper inactivation of infectious agents before disposal.
**Important Notes:**
- Substrate solutions must be used before they become contaminated or discolored.
- Avoid contact with heat or flame when handling Chromogen B, which contains 20% acetone.
- Always read the entire user manual before beginning the assay.
This kit is ideal for researchers studying cell adhesion, immune responses, and inflammatory processes involving ICAM-3. Proper handling and adherence to instructions will ensure reliable and reproducible results.