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Human 25- Dihydroxy vitamin D;25-dihydroxyvitamin D(3);25-OH-D ELISA Kit
Principle
This ELISA kit uses Sandwich-ELISA as the method. The Microelisa stripplate provided in this kit has been pre-coated with an antibody specific to 25- Dihydroxy vitamin D (25-OH-D) . Standards or samples are added to the appropriate Microelisa stripplate wells and combined to the specific antibody. Then a Horseradish Peroxidase (HRP)- conjugated antibody specific for 25- Dihydroxy vitamin D (25-OH-D) is added to each Microelisa stripplate well and incubated. Free components are washed away. The TMB substrate solution is added to each well. Only those wells that contain 25- Dihydroxy vitamin D (25-OH-D)and HRP conjugated 25- Dihydroxy vitamin D (25-OH-D)antibody will appear blue in color and then turn yellow after the addition of the stop solution. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm. The OD value is proportional to the concentration of 25- Dihydroxy vitamin D (25-OH-D) . You can calculate the concentration of 25- Dihydroxy vitamin D (25-OH-D)in the samples by comparing the OD of the samples to the standard curve.
Materials provided with the kit
| Materials provided with the kit | 48 determinations | 96 determinations | Storage | |
| 1 | User manual | 1 | 1 | R.T. |
| 2 | Closure plate membrane | 2 | 2 | R.T. |
| 3 | Sealed bags | 1 | 1 | R.T. |
| 4 | Microelisa stripplate | 1 | 1 | 2-8℃ |
| 5 | Standard 90ng/ml | 0.5ml×1 bottle | 0.5ml×1 bottle | 2-8℃ |
| 6 | Standard diluent | 1.5ml×1 bottle | 1.5ml×1 bottle | 2-8℃ |
| 7 | HRP-Conjugate reagent | 3ml×1 bottle | 6ml×1 bottle | 2-8℃ |
| 8 | Sample diluent | 3ml×1 bottle | 6ml×1 bottle | 2-8℃ |
| 9 | Chromogen Solution A | 3ml×1 bottle | 6ml×1 bottle | 2-8℃ |
| 10 | Chromogen Solution B | 3ml×1 bottle | 6ml×1 bottle | 2-8℃ |
| 11 | Stop Solution | 3ml×1 bottle | 6ml×1 bottle | 2-8℃ |
| 12 | Wash solution | 20ml (20X)×1bottle | 20ml (30X)×1bottle | 2-8℃ |
Notes:Standard curve should be included in every assay. Replicate wells are recommended. If the OD value of the sample is greater than the first well of standards, please dilute the sample (n times) before test. When calculating the original 25-OH-D concentration, please multiply the total dilution factor (XnX5).
Calculation of Results
Known concentrations of Human 25- Dihydroxy vitamin D (25-OH-D) Standard and its corresponding reading OD is plotted on the log scale (x-axis) and the log scale (y-axis) respectively. The concentration of Human 25- Dihydroxy vitamin D (25-OH-D) in sample is determined by plotting the sample’s O.D. on the Y-axis. The original concentration is calculated by multiplying the dilution factor.
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Human 25- Dihydroxy vitamin D (25-OH-D) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Human 25- Dihydroxy vitamin D (25-OH-D) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%
Assay range
3 ng/ml -80 ng/ml
