Human Vitamin D3;VD3 ELISA Kit

• Expiration date:six months .
• Storage: 2-8℃.
• Our Human Vitamin D3 (VD3) ELISA kit is to assay Vitamin D3 (VD3) levels in Human serum, plasma, culture media or any biological fluid.

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 Vitamin D3 (VD3) . 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 Vitamin D3 (VD3) 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 Vitamin D3 (VD3) and HRP conjugated Vitamin D3 (VD3) 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 Vitamin D3 (VD3) . You can calculate the concentration of Vitamin D3 (VD3) in the samples by comparing the OD of the samples to the standard curve.

Materials provided with the kit

• Dilution of Standards

Ten wells are set for standards in a Microelisa stripplate. In Well 1 and Well 2, 100μl Standard solution and 50μl Standard Dilution buffer are added and mixed well. In Well 3 and Well 4, 100μl solution from Well 1 and Well 2 are added respectively. Then 50μl Standard Dilution buffer are added and mixed well. 50μl solution is discarded from Well 3 and Well 4. In Well 5 and Well 6, 50μl solution from Well 3 and Well 4 are added respectively. Then 50μl Standard Dilution buffer are added and mixed well. In Well 7 and Well 8, 50μl solution from Well 5 and Well 6 are added respectively. Then 50μl Standard Dilution buffer are added and mixed well. In Well 9 and Well 10, 50μl solution from Well 7 and Well 8 are added respectively. Then 50μl Standard Dilution buffer are added and mixed well. 50μl solution is discarded from Well 9 and Well 10. After dilution, the total volume in all the wells are 50μl and the concentrations are 30 ng/ml, 20 ng/ml, 10 ng/ml, 5 ng/ml and 2.5 ng/ml, respectively.

Calculation of Results

  Known concentrations of Human Vitamin D3 (VD3) 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 Vitamin D3 (VD3) 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 Vitamin D3 (VD3) were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Human Vitamin D3 (VD3)were tested on 3 different plates, 8 replicates in each plate.

CV(%) = SD/meanX100

Intra-Assay: CV<10%

Inter-Assay: CV<12%

Assay range

0.8 ng/ml -36 ng/ml

Sensitivity:

0.3 ng/ml

• Store the kit at 4°C upon receipt.The kit should be equilibrated to room temperature before the assay. Remove any unneeded strips from Human Vitamin D3 (VD3) Antibody-Coated plate, reseal them in zip-lock foil and keep at 4°C.
• 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 Vitamin D3 (VD3) concentration, please multiply the total dilution factor (XnX5).