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Manual Download| Product Name | 25-OH-D(25 Hydroxy Vitamin D) ELISA Kit | Species | |
| Uniprot ID | N/A | Alternative Names | 25(OH)D |
| Detection method | Competitive | Sensitivity | 0.91 ng/mL |
| Standard | 200ng/mL | Detection Range | 3.13-200ng/mL |
| Sample type | Serum, Plasma, Tissue homogenate and Other biological samples；Sample Volume=50μL | ||
| Reaction time | 1.5H | Research Area | Cardiovascular，Signaling transduction，Neuroscience，Cancer，Metabolism |
| Test principle | This ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with 25-OH-D. During the reaction, 25-OH-D in the sample or standard competes with a fixed amount of 25-OH-D on the solid phase supporter for sites on the Biotinylated Detection Ab specific to 25-OH-D. Excess conjugate and unbound sample or standard are washed away, and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns from blue to yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of 25-OH-D in tested samples can be calculated by comparing the OD of the samples to the standard curve. | ||
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
| (ng/mL) | OD |
|---|---|
| 200.00 | 0.234 |
| 100.00 | 0.336 |
| 50.00 | 0.612 |
| 25.00 | 0.979 |
| 12.50 | 1.292 |
| 6.25 | 1.554 |
| 3.13 | 2.022 |
| 0.00 | 2.663 |
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level were tested on 3 different plates, 20 replicates in each plate, respectively.
| Intra-assay Precision | Inter-assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean(ng/mL) | 6.152 | 35.857 | 84.1 | 5.241 | 20.486 | 92.6 |
| Standard deviation | 0.405 | 1.259 | 3.078 | 0.409 | 0.85 | 3.297 |
| C V (%) | 6.58 | 3.51 | 3.66 | 7.8 | 4.15 | 3.56 |
The recovery of spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range(%) | Average Recovery(%) |
|---|---|---|
| Serum (n=8) | 88-94 | 91 |
| EDTA plasma (n=8) | 87-95 | 92 |
| Cell culture media (n=8) | 90-97 | 94 |
Samples were spiked with high concentrations of target proteins and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
|---|---|---|---|---|
| 1:2 | Range (%) | 93-100 | 96-104 | 88-95 |
| Average (%) | 97 | 99 | 92 | |
| 1:4 | Range (%) | 95-104 | 90-99 | 98-107 |
| Average (%) | 99 | 94 | 102 | |
| 1:8 | Range (%) | 89-97 | 89-96 | 89-96 |
| Average (%) | 94 | 93 | 92 | |
| 1:16 | Range (%) | 94-103 | 87-97 | 97-105 |
| Average (%) | 99 | 92 | 100 |
