Vitamin D (25OH) (Free)

This ELISA kit applies to the in vitro quantitative determination of Vitamin D (25OH) (Free) concentrations in serum, plasma and other biological fluids.

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antigen specific to Vitamin D (25OH) (Free). Standards or samples are added to the micro ELISA plate wells and combined with the specific antigen. Then a biotinylated detection antigen specific for Vitamin D (25OH) (Free) and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Vitamin D (25OH) (Free), biotinylated detection antigen and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Vitamin D (25OH) (Free). You can calculate the concentration of Vitamin D (25OH) (Free) in the samples 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.

Intra-assay Precision (Precision within an assay):
3 samples with low, middle,high concentration of Vitamin D (25OH) (Free). And the sample of Vitamin D (25OH) (Free) were tested 30 times on one plate, respectively.
Inter-assay Precision (Precision between assays):
3 samples with low, middle,high concentration of Vitamin D (25OH) (Free). And the sample of Vitamin D (25OH) (Free) were tested on 3 different plates, 30 replicates in each plate.

Matrices listed below were spiked with certain level of Vitamin D (25OH) (Free) and the recovery rates were calculated by comparing the measured value to the expected amount of Vitamin D (25OH) (Free) in samples.

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Vitamin D (25OH) (Free) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

The stability of Vitamin D (25OH) (Free) is determined by the loss rate of activity. The loss rate of Vitamin D (25OH) (Free) is less than 5% within the expiration date under appropriate storage condition. To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Microplate reader with 450 nm wavelength filter
High-precision transfer pipette, EP tubes and disposable pipette tips
Incubator capable of maintaining 37℃
Deionized or distilled water
Absorbent paper
Loading slot for Wash Buffer

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 1 hours at 37°C;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.