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Leucine Aminopeptidase (LAP) Activity Assay Kit
SKU: E-BC-K568-M-96
Leucine Aminopeptidase (LAP) Activity Assay Kit
| SKU # | E-BC-K568-M |
| Detection Instrument | Microplate reader (405 nm) |
| Detection method | Colorimetric method |
Product Details
Properties
| Synonyms | LAP |
| Sample type | Serum, plasma, animal tissue |
| Sensitivity | 5.2 U/L |
| Detection range | 5.2-201.8 U/L |
| Detection Method | Colorimetric method |
| Assay type | Enzyme Activity |
| Assay time | 25 min |
| Precision | Average inter-assay CV: 6.400% | Average intra-assay CV: 4.100% |
| Other instruments required | Micropipettor, Water bath, Centrifuge, Incubator, Vortex mixer |
| Other reagents required |
Acetone |
| 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 ( 5.2-201.8 U/L).
The recommended dilution factor for different samples is as follows (for reference only):
| Sample type | Dilution factor |
| Human serum | 1 |
| Dog serum | 1 |
| Rat serum | 1 |
| Cynomolgus monkey serum | 1 |
| 10% Rat spleen tissue homogenate | 1 |
| 10% Rat liver tissue homogenate | 1 |
| 10% Rat kidney tissue homogenate | 1 |
| 10% Rat lung tissue homogenate | 1 |
Note: The diluent is reagent 1.
Detection Principle
LAP can catalyze the substrate L-leucine-4-nitroaniline to produce p-nitroaniline, which has the maximum absorption peak at the wavelength of 405 nm. The enzyme activity of LAP can be calculated by measuring the increasing OD value of the system.
Kit Components & Storage
| Item | Component | Size (96 T) | Storage |
| Reagent 1 | Extracting Solution | 60 mL ×2 vials | 2-8°C, 12 months |
| Reagent 2 | Substrate | Powder × 2 vials | 2-8°C, 12 months, shading light |
| Reagent 3 | p-Nitroaniline Standard | Powder ×1 vial | 2-8°C, 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 (U/L) | 12.5 | 65.8 | 175.0 |
| %CV | 4.5 | 4.0 | 3.8 |
Inter-assay Precision
Three human serum samples were assayed 17 times in duplicate by three operators to determine precision between assays.
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (U/L) | 12.5 | 65.8 | 175.0 |
| %CV | 6.2 | 6.0 | 7.0 |
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 99%.
| Standard 1 | Standard 2 | Standard 3 | |
| Expected Conc. (mmol/L) | 0.25 | 0.55 | 0.82 |
| Observed Conc. (mmol/L) | 0.3 | 0.5 | 0.8 |
| Recovery rate (%) | 101 | 99 | 97 |
Sensitivity
The analytical sensitivity of the assay is 5.2 U/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 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 (μmol/L) | 0.0 | 0.2 | 0.3 | 0.4 | 0.6 | 0.8 | 0.9 | 1.0 |
| Average OD | 0.056 | 0.111 | 0.137 | 0.158 | 0.211 | 0.269 | 0.304 | 0.328 |
| Absoluted OD | 0.000 | 0.055 | 0.081 | 0.102 | 0.155 | 0.213 | 0.248 | 0.272 |