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**Human ICTP ELISA Kit – For the Quantitative In Vitro Determination of Human Carboxy-terminal Telopeptide Type I (ICTP) in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
*For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.*
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### **INTENDED USE AND TEST PRINCIPLE**
This Human ICTP ELISA Kit is specifically designed for the quantitative determination of carboxy-terminal telopeptide type I (ICTP) in various biological samples, including serum, plasma, cerebrospinal fluid, tissue homogenates, and other body fluids. It is intended solely for laboratory research purposes and should not be used for diagnostic or therapeutic applications.
The assay is based on a sandwich ELISA format. A microtiter plate coated with specific antibodies is used to capture ICTP from the sample. A horseradish peroxidase (HRP)-conjugated detection antibody is then added, followed by a chromogenic substrate that produces a color change. The reaction is stopped, and the optical density (OD) at 450 nm is measured using a spectrophotometer. The intensity of the color is directly proportional to the concentration of ICTP in the sample.
A set of calibration standards is included in the kit to generate a standard curve. This curve allows for accurate quantification of ICTP levels in unknown samples by comparing their OD values to those of the standards.
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### **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 before centrifugation at 2000×g for 20 minutes. Remove the serum and analyze 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. Store at -20°C and avoid repeated freezing and thawing.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates. Analyze immediately or store at -20°C. Avoid repeated freeze-thaw cycles.
*Note: Ensure adequate centrifugation and avoid hemolysis or contamination with cellular debris.*
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### **MATERIALS REQUIRED BUT NOT SUPPLIED**
1. Incubator set 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
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### **REAGENTS PROVIDED**
| Reagent | 96 Determinations | 48 Determinations |
|---------|-------------------|-------------------|
| Microplate (12×8 strips) | 1 | 1 |
| Standards (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 |
*Standard concentrations: 800, 400, 200, 100, 50, 25 pg/mL.*
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### **INSTRUCTIONS FOR USE**
1. Bring all reagents to room temperature (20–25°C) before use. Do not use water baths for thawing.
2. Prepare the 1X Wash Solution by diluting 1 volume of 20X Wash Solution with 19 volumes of distilled or deionized water. Store at 2–8°C for up to one month.
3. Add 50 µL of standards or samples in duplicate to the microplate wells. Do not add anything to the blank well.
4. Add 100 µL of HRP-Conjugate Reagent to each well except the blank. Cover with an adhesive strip and incubate for 60 minutes at 37°C.
5. Wash the plate 4 times using either manual or automated washing methods. Ensure complete removal of unbound conjugate.
6. 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.
7. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. If the color appears green or inconsistent, gently mix the plate.
8. Measure the OD at 450 nm using a microplate reader. Subtract the blank OD from all readings.
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### **DATA ANALYSIS**
1. Plot the average OD values of the standards against their concentrations to create a standard curve.
2. Calculate the mean OD for each sample and subtract the blank value.
3. Determine the ICTP concentration by interpolating the sample OD on the standard curve.
4. Intra-assay and inter-assay CV% are less than 15%.
5. Assay range: 25 pg/mL to 800 pg/mL.
6. Sensitivity: <1.0 pg/mL.
7. Cross-reactivity: No significant cross-reactivity with other proteins was observed.
8. Storage: Store unused kits at 2–8°C for frequent use or at -20°C for long-term storage (up to 6 months).
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### **SAFETY AND DISPOSAL**
- Handle all samples as potentially hazardous. Wear gloves during the procedure.
- Waste materials must be inactivated for at least 30 minutes before disposal.
- Substrate solutions may be contaminated; do not use if discolored.
- Chromogen B contains 20% acetone—keep away from heat and flame.
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**Please read and follow all instructions carefully. This kit is not intended for use in clinical diagnostics.**
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