Species:
Universal
Alternative Name:
Cholecalciferol; Toxiferol; Activated 7-Dehydrocholesterol
Assay Type:
Competitive
unit:
ng/mL
Standard:
400 ng/mL
Sensitivity:
2.14 ng/mL
Detection Range:
6.25-400 ng/mL
Assay Time:
1.5H
Sample Type:
Serum, Plasma and Other biological fluids
Research:
Metabolic pathway;Reproductive science;Nutrition metabolism;Bone metabolism;
Test Principle:
This ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with VD3. Standards or samples are added to the appropriate micro ELISA plate wells then with a biotin-conjugated antibody specific to VD3. Next,Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of VD3 in the samples is then determined by comparing the OD of the samples to the standard curve.
| ng/mL | OD | Corrected |
|---|---|---|
| 400 | 0.258 | |
| 200 | 0.421 | |
| 100 | 0.661 | |
| 50 | 0.978 | |
| 25 | 1.271 | |
| 12.5 | 1.591 | |
| 6.25 | 1.861 | |
| 0 | 2.683 |
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) | 7.87 | 16.81 | 112.36 | 8.2 | 12.58 | 139.86 |
| Standard deviation | 0.84 | 1.85 | 5.8 | 1.04 | 2.31 | 7.22 |
| C V (%) | 5.18 | 6.22 | 3.37 | 5.73 | 5.38 | 4.14 |
| Sample Type | Range(%) | Average Recovery(%) |
|---|---|---|
| Serum (n=8) | 92-105 | 98 |
| EDTA plasma (n=8) | 84-96 | 90 |
| Cell culture media (n=8) | 80-95 | 87 |
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
|---|---|---|---|---|
| 1:2 | Range (%) | 86-93 | 93-102 | 91-104 |
| Average (%) | 91 | 98 | 92 | |
| 1:4 | Range (%) | 91-105 | 93-102 | 89-97 |
| Average (%) | 96 | 101 | 93 | |
| 1:8 | Range (%) | 87-98 | 93-106 | 93-102 |
| Average (%) | 90 | 102 | 97 | |
| 1:16 | Range (%) | 89-94 | 87-101 | 81-96 |
| Average (%) | 94 | 90 | 88 |