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Total Cholesterol and Cholesteryl Ester Fluorometric Assay Kit
SKU: E-BC-F032-96
Total Cholesterol and Cholesteryl Ester Fluorometric Assay Kit
| SKU # | E-BC-F032 |
| Detection Instrument | Fluorescence microplate reader (Ex/Em=535 nm/590 nm) |
| Detection Method | Fluorescence method |
Product Details
Properties
| Sample Type | Serum, plasma, animal tissue, cells |
| Sensitivity | 0.12 μmol/L |
| Detection Range | 0.12-30 μmol/L |
| Detection Method | Fluorescence method |
| Assay Time | 20 min |
| Precision | Inter-assay CV: 7.3% | intra-assay CV: 1.7% |
| Storage | This product can be stored at -20°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 serum | 100-300 |
| Rat serum | 100-300 |
| Mouse plasma | 100-300 |
| Rabbit serum | 100-300 |
| 10% Rat liver tissue homogenate | 50-150 |
| 10% Mouse kidney tissue homogenate | 50-200 |
| 10% Rat brain tissue homogenate | 200-400 |
| 10% Rat spleen tissue homogenate | 50-200 |
| Jukat cells | 20-50 |
The diluent is extracting solution. For the dilution of other sample types, please do pretest to confirm the dilution factor.
Detection Principle
Total Cholesterol (TC) includes free cholesterol (FC) and cholesteryl esters (CE). Cholesterol ester can be hydrolyzed by cholesterol esterase to produce cholesterol and free fatty acid. Cholesterol is oxidized by cholesterol oxidase to produce △4-cholestenone and hydrogen peroxide. In the presence of the enzyme and probe, hydrogen peroxide can be catalyzed to produce the fluorescence substrate. The fluorescence intensity at the excitation wavelength of 535 nm and emission wavelength of 590 nm is proportional to the cholesterol concentration.
Kit Components & Storage
| Item | Component | Size 1(48 T) | Size 2(96 T) | Storage |
| Reagent 1 | Buffer Solution | 30 mL × 1 vial | 60 mL × 1 vial | -20℃, 12 months |
| Reagent 2 | Substrate | 0.06 mL × 1 vial | 0.12 mL × 1 vial | -20℃, 12 months, shading light |
| Reagent 3 | Enzyme Reagent 1 | 0.3 mL × 1 vial | 0.3 mL × 1 vial | -20℃, 12 months, shading light |
| Reagent 4 | Enzyme Reagent 2 | 0.3 mL × 1 vial | 0.3 mL × 1 vial | -20℃, 12 months |
| Reagent 5 | 5.17 mmol/L Cholesterol Standard Solution |
0.2 mL × 1 vial | 0.2 mL × 1 vial | -20℃, 12 months |
| Reagent 6 | Extracting Solution | 30 mL × 1 vial | 60 mL × 1 vial | -20℃, 12 months, shading light |
| Black 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 (μmol/L) | 1.50 | 14.00 | 25.00 |
| %CV | 1.8 | 1.6 | 1.7 |
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 (μmol/L) | 1.50 | 14.00 | 25.00 |
| %CV | 7.9 | 6.8 | 7.2 |
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 96%
| Standard 1 | Standard 2 | Standard 3 | |
| Expected Conc.(μmol/L) | 2.5 | 12 | 23 |
| Observed Conc. (μmol/L) | 2.5 | 11.4 | 21.9 |
| Recovery rate (%) | 98 | 95 | 95 |
Sensitivity
The analytical sensitivity of the assay is 0.12 μmol/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:
As the fluorescence 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 (μmol/L) | 0 | 2 | 5 | 10 | 15 | 20 | 25 | 30 |
| Fluorescence value | 210 | 741 | 1834 | 3334 | 4630 | 6141 | 7432 | 8394 |
| 216 | 786 | 1828 | 3346 | 4745 | 6165 | 7482 | 8524 | |
| Average fluorescence value | 213 | 764 | 1831 | 3340 | 4688 | 6153 | 7457 | 8459 |
| Absoluted fluorescence value | 0 | 550 | 1618 | 3127 | 4475 | 5940 | 7244 | 8246 |