25-Hydroxyvitamin D3 (HVD3) BioAssay™ ELISA Kit
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The 25-Hydroxy Vitamin D3 (HVD3) ELISA Kit is a competitive inhibition immunoassay for the in vitro quantitative measurement of HVD3 in serum, plasma and other biological fluids.
Intra-Assay CV: <10%
Inter-Assay CV: <12%
This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific for HVD3 has been pre-coated onto a microplate. A competitive inhibition reaction occurs between biotin-labeled HVD3 and unlabeled HVD3 (standards or samples) with the pre-coated antibody specific for HVD3. 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 inversely proportional to the concentration of HVD3 in the sample. After addition of the substrate solution, the intensity of color developed is inversely proportional to the concentration of HVD3 in the sample.
*023031A: Microtiter Plate, 96 wells, Pre-coated, ready to use
*023031B: Standard, 2x1vial
023031C: Standard Diluent, 1x20ml
*023031D: Detection Reagent A, 1x1vial
*023031E: Detection Reagent B, 1x120ul
023031F: Assay Diluent A, 1x12ml
023031G: Assay Diluent B, 1x12ml
023031H: TMB Substrate, 1x9ml
023031K: Stop Solution, 1x6ml
023031L: Wash Buffer, 30X, 1x20ml
023031M: Reagent Diluent, 1x300ul
The Stop Solution (023031K) suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
Storage and Stability:
Store *023031A, *023031B, *023031D and *023031E at -20°C. Store all the other components at 4°C. Unused kit is stable for 6 months. Once kit components are opened, it is highly recommended to use remaining reagents within 1 month provided this is within the expiration date of the kit. For maximum recovery of product, centrifuge the original vials after thawing and prior to removing the cap.
Materials Required But Not Supplied:
1. Microplate reader with 450 ± 10nm filter.
2. Precision single or multi-channel pipettes and disposable tips.
3. Eppendorf Tubes for diluting samples.
4. Deionized or distilled water.
5. Absorbent paper for blotting the microtiter plate.
6. Container for Wash Solution
Sample Preparation and Storage:
Allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000xg. Assay freshly prepared serum immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.
Collect plasma using EDTA or heparin as anticoagulant. Centrifuge samples for 15 minutes at 1000xg at 2-8°C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.
Other Biological Fluids:
Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquots at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C ( 1 month) or -80°C ( 2 months) to avoid loss of bioactivity and contamination.
2. Sample hemolysis will influence the results so hemolyzed specimens should not be used.
3. When performing the assay, bring samples to room temperature.
Assay Procedure Summary:
1. Prepare all reagents, samples and standards.
2. Add 50ul standard or sample to each well, then add 50ul prepared Detection Reagent A immediately. Shake and mix. Incubate 1 hour at 37°C.
3. Aspirate and wash 3 times.
4. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37°C.
5. Aspirate and wash 5 times.
6. Add 90ul Substrate Solution. Incubate 15-25 minutes at 37°C.
7. Add 50ul Stop Solution. Read absorbance at 450nm immediately.
Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.