ELISA Kit for 25-Hydroxyvitamin D3 (HVD3)

This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to HVD3 has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled HVD3 analogues and unlabeled antigen (Standards or samples) with the pre-coated antibody. After incubation the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of HVD3 in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of HVD3 in the sample.

1. Prepare all reagents, samples and standards; <br/> 2. Add 50&micro; L standard or sample to each well.<br/> &nbsp; &nbsp; &nbsp; &nbsp; And then add 50&micro; L prepared Detection Reagent A immediately.<br/> &nbsp; &nbsp; &nbsp; &nbsp; Shake and mix. Incubate 1 hour at 37&deg; C; <br/> 3. Aspirate and wash 3 times; <br/> 4. Add 100&micro; L prepared Detection Reagent B. Incubate 30 minutes at 37&deg; C; <br/> 5. Aspirate and wash 5 times; <br/> 6. Add 90&micro; L Substrate Solution. Incubate 10-20 minutes at 37&deg; C; <br/> 7. Add 50&micro; L Stop Solution. Read at 450 nm immediately.<br/>

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level 25-Hydroxyvitamin D3 (HVD3) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level 25-Hydroxyvitamin D3 (HVD3) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV Inter-Assay: CV Inter-Assay: CV

The stability of kit is determined by the loss rate of activity. The loss rate of this kit 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.

This assay has high sensitivity and excellent specificity for detection of 25-Hydroxyvitamin D3 (HVD3) . No significant cross-reactivity or interference between 25-Hydroxyvitamin D3 (HVD3) and analogues was observed.