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Free Cholesterol (FC) Colorimetric Assay Kit
SKU: E-BC-K004-M-500
Free Cholesterol (FC) Colorimetric Assay Kit
| SKU # | E-BC-K004-M |
| Detection Instrument | Microplate reader(495-525 nm, optimum wavelength: 510 nm) |
| Detection Method | Colorimetric method |
Product Details
Properties
| Synonyms | FC |
| Sample Type | Serum, plasma, tissue |
| Sensitivity | 0.07 mmol/L |
| Detection Range | 0.07-24 mmol/L |
| Detection Method | Colorimetric method |
| Assay Time | 20 min |
| Precision | Inter-assay CV: 5.2% | intra-assay CV: 1.9% |
| Other Reagents Required | Normal saline (0.9% NaCl), PBS (0.01 M, pH 7.4), Isopropanol(AR) |
| Storage | This product can be stored at 2~8°C for 12 months with shading light. |
| Valid period | 12 months |
Dilution Of Sample
The recommended dilution factor for different samples is as follows (for reference only):
| Sample type | Dilution factor |
|---|---|
| Human plasma | 1 |
| Human serum | 1 |
| Rat serum | 1 |
| Mouse serum | 1 |
| Rabbit serum | 1 |
| 10% Rat kidney tissue homogenate | 1 |
The diluent is normal saline (0.85% NaCl) or PBS (0.01 M, pH 7.4). For the dilution of other sample types, please do pretest to confirm the dilution factor.
Detection Principle
Free cholesterol produces 4-cholestenone and hydrogen peroxide under the oxidation of cholesterol oxidase. In the presence of 4-aminoamylpyridine and phenol, peroxidase catalyze hydrogen peroxide to form red quinone compounds of benzoquinone imine phenizone. The color depth of the generated quinones is directly proportional to the cholesterol content.
Kit Components & Storage
| Item | Component | Size 1(48 T) | Size 2(96 T) | Storage |
| Reagent 1 | Enzyme Working Solution |
15 mL ×1 vial | 30 mL ×1 vial | 2-8°C, 12 months, shading light |
| Reagent 2 | 5.17 mM Cholesterol Standard |
0.2 mL ×1 vial | 0.2 mL × 1 vial | 2-8℃, 12 months |
| Microplate | 96 wells | No requirement | ||
| Plate Sealer | 2 pieces | |||
Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as not to obtain sufficient amount of reagents.
Technical Data:
Parameter:
Intra-assay Precision
Three human serum samples were assayed in replicates of 20 to determine precision within an assay. (CV = Coefficient of Variation)
| Parameters | Sample 1 | Sample 2 | Sample 3 |
|
Mean (mmol/L) |
1.00 | 10.00 | 17.00 |
| %CV | 2.5 | 1.9 | 1.3 |
Inter-assay Precision
Three human serum samples were assayed 20 times in duplicate by three operators to determine precision between assays.
| Parameters | Sample 1 | Sample 2 | Sample 3 |
|
Mean (mmol/L) |
1.00 | 10.00 | 17.00 |
| %CV | 5.7 | 4.3 | 5.6 |
Recovery
Take three samples of high concentration, middle concentration and low concentration to test the samples of each concentration for 6 times parallelly to get the average recovery rate of 100%.
| Sample 1 | Sample 2 | Sample 3 | |
| Expected Conc. (mmol/L) | 8 | 18 | 20 |
| Observed Conc. (mmol/L) | 7.9 | 17.6 | 18.8 |
| Recovery rate (%) | 99 | 98 | 94 |
Sensitivity
The analytical sensitivity of the assay is 0.07 mmol/L. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.
Standard Curve:
(It doesn't need to prepare the standard curve for this kit and the provided standard curve is for reference only)
As the OD value of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique or temperature effects), so the standard curve and data are provided as below for reference only.
| Concentration (mmol/L) | 0 | 4 | 6 | 8 | 12 | 16 | 20 | 24 |
| OD value | 0.078 | 0.420 | 0.567 | 0.734 | 1.082 | 1.380 | 1.767 | 2.122 |
| 0.077 | 0.436 | 0.581 | 0.728 | 1.102 | 1.436 | 1.746 | 2.092 | |
| Average OD | 0.078 | 0.428 | 0.574 | 0.731 | 1.092 | 1.408 | 1.756 | 2.107 |
| Absoluted OD | 0.000 | 0.351 | 0.497 | 0.653 | 1.014 | 1.331 | 1.679 | 2.029 |