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|Introduction||25 Hydroxyvitamin D (25(OH)D, also known as Calcidiol and Calcifediol) is an intermediate chemical produced during the activation of vitamin D. It is produced from cholecalciferol by cholecalciferol 25-hydroxylase (CYP2R1) in the endoplasmic reticulum, and is later converted into calcitriol by 25-hydroxyvitamin D3 1-α-hydroxylase (CYP27B1). Low serum concentrations of calcidiol can be indicative of osteoporosis, vitamin malabsorption, or chronic kidney disease.|
|Target||25-Hydroxyvitamin D3 (Calcidiol)|
|Reactivity||General (All species)|
|Recommended dilutions||Optimal dilutions/concentrations should be determined by the end user.|
|Storage||Shipped at 4 °C. Upon receipt, store the kit according to the storage instruction in the kit's manual.|
|Validity||The validity for this kit is 6 months.|
|Stability||The stability of the kit is determined by the rate of activity loss. The loss rate is less than 5% within the expiration date under appropriate storage conditions. To minimize performance fluctuations, operation procedures and lab conditions should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same user throughout.|
|Test Range||3.125 ng/ml - 200 ng/ml|
|Sample Type||Serum, plasma, tissue homogenates, cell lysates and other biological fluids.|
|Assay Principle||This kit is based on competitive enzyme-linked immuno-sorbent assay technology. An antigen is pre-coated onto a 96-well plate. Standards, test samples, and biotin-conjugated reagent are added to the wells and incubated. A competitive inhibition reaction takes place between the the pre-coated Calcidiol and the Calcidiol in the sample with the biotin-labelled antibody. The HRP-conjugated reagent is then added, and the whole plate is incubated. Unbound conjugates are removed using wash buffer at each stage. TMB substrate is used to quantify the HRP enzymatic reaction. After TMB substrate is added, only wells that contain sufficient Calcidiol will produce a blue coloured product, which then changes to yellow after adding the acidic stop solution. The intensity of the color yellow is inversely proportional to the Calcidiol amount bound on the plate. The OD is measured spectrophotometrically at 450 nm in a microplate reader, from which the concentration of Calcidiol can be calculated.|
|Kit Components||The kit components listed are for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product.
|Material Required But Not Provided||
|Reagent Preparation||This procedure is provided for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product.
|Assay Procedure||This procedure is provided for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product.
|Protocol||This procedure is provided for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product.
|Results Calculation||This assay is competitive, therefore there is an inverse correlation between Calcidiol concentration in the sample and the absorbance measured. Create a graph with the log of the standard concentration (y-axis) and average absorbance measured (x-axis). Apply a best fit trendline through the standard points. The Calcidiol concentration of the samples can be interpolated from the standard curve.|
|Assay Precision||Intra-assay Precision (Precision within an assay): 3 samples with low, medium and high levels of 25-Hydroxyvitamin D3 (Calcidiol) were were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, medium and high levels of 25-Hydroxyvitamin D3 (Calcidiol) were tested on 3 different plates, 8 replicates in each plate.
CV (%) = (Standard Deviation / mean) × 100
|Availability||Shipped within 5-12 working days.|
|Note||This product is for research use only. The range and sensitivity is subject to change. Please contact us for the latest product information. For accurate results, sample concentrations must be diluted to mid-range of the kit. If you require a specific range, please contact us in advance or write your request in your order comments. Please note that our ELISA and CLIA kits are optimised for detection of native samples, rather than recombinant proteins. We are unable to guarantee detection of recombinant proteins, as they may have different sequences or tertiary structures to the native protein.|
|Plate coated with||Antigen|