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Alkaline Phosphatase (ALP) Activity Assay Kit
SKU: E-BC-K091-M-500
Alkaline Phosphatase (ALP) Activity Assay Kit
| SKU # | E-BC-K091-M |
| Detection Instrument | Microplate reader (500-530 nm, optimum wavelength: 520 nm) |
| Detection Method | Colorimetric method |
Product Details
Properties
| Synonyms | ALP |
| Sample Type | Serum, plasma, tissue, cells |
| Sensitivity | 0.13 King unit/100 mL |
| Detection Range | 0.13-50 King unit/100 mL |
| Detection Method | Colorimetric method |
| Assay type | Enzyme Activity |
| Assay time | 30 min |
| Precision | Average inter-assay CV: 8.500% | Average intra-assay CV: 5.100% |
| Other instruments required | Micropipettor, Centrifuge, Incubator, Vortex mixer |
| Other reagents required | Normal saline (0.9% NaCl), PBS (0.01 M, pH 7.4) |
| Storage | 2-8℃ |
| Valid period | 12 months |
Dilution of Sample
It is recommended to take 2~3 samples with expected large difference to do pre-experiment before formal experiment and dilute the sample according to the result of the pre-experiment and the detection range (0.13-50 King unit/100 mL).
The recommended dilution factor for different samples is as follows (for reference only):
| Sample type | Dilution factor |
| Human plasma | 1 |
| Human urine | 1 |
| Rat serum | 1 |
| Cells culture supernatant | 1 |
| 10% Mouse kidney tissue homogenate | 30-50 |
| 0% Mouse liver tissue homogenate | 1 |
| 10% Mouse brain tissue homogenate | 1 |
| HepG2 cells | 1 |
Note: The diluent is normal saline (0.9% NaCl), PBS (0.01 M, pH 7.4).
Detection Principle
Alkaline phosphatase decompose benzene disodium phosphate to produce free phenol and phosphoric acid. Phenol react with 4-aminopyrline in alkaline solution and oxidizes with potassium ferricyanide to form red quinone derivative. The enzyme activity can be calculated indirectly by measuring the OD value.
Kit Components & Storage
| Item | Component | Size 1(96 T) | Size 2 (500 Assays) | Storage |
| Reagent 1 | Buffer Solution | 3 mL ×1 vial | 15 mL ×1 vial | 2-8℃, 12 months, shading light |
| Reagent 2 | Substrate Solution | 3 mL × 1 vial | 15 mL ×1 vial | 2-8℃, 12 months, shading light |
| Reagent 3 | Chromogenic Agent | 18 mL × 1 vial | 50 mL × 2 vials | 2-8℃, 12 months, shading light |
| Reagent 4 | 0.5 mg/mL Phenol Standard | 1.5 mL × 1 vial | 5 mL × 1 vial | 2-8℃, 12 months, shading light |
| 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 (King unit/100 mL) | 1.40 | 24.60 | 41.50 |
| %CV | 5.4 | 4.9 | 5.0 |
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 (King unit/100 mL) | 1.40 | 24.60 | 41.50 |
| %CV | 8.7 | 8.0 | 8.8 |
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 94%.
| Sample 1 | Sample 2 | Sample 3 | |
| Expected Conc. (mg/mL) | 0.036 | 0.17 | 0.38 |
| Observed Conc. (mg/mL) | 0.0 | 0.2 | 0.4 |
| Recovery rate (%) | 95 | 93 | 94 |
Sensitivity
The analytical sensitivity of the assay is 0.13 King unit/100 mL. 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 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 (mg/mL) | 0 | 0.025 | 0.05 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 |
| Average OD | 0.054 | 0.135 | 0.215 | 0.371 | 0.664 | 0.965 | 1.265 | 1.563 |
| Absoluted OD | 0 | 0.081 | 0.161 | 0.317 | 0.610 | 0.911 | 1.211 | 1.509 |