The primary synthesis of functional vitamin D begins in the skin, where a cholesterol by-product acted upon by UV light forms vitamin D3. Vitamin D3 is carried through the bloodstream, attached to the vitamin D binding protein, to the liver where it is converted to 25-hydroxy vitamin D3 (25(OH)D3). This product is then transported to the kidney where it is further converted to 1,25-dihydroxycholecalciferol (1,25(OH)2D3), the biologically active form. Active 1,25(OH)2D3 is very short-lived and is rapidly metabolized to the deactivated forms 24,25(OH)2D3 and 1,24,25(OH)3D3. Vitamin D Immunoassay: Accurate Vitamin D measurement in plasma or serum Cayman’s Vitamin D ELISA Kit is a competitive assay that can be used for Vitamin D quantification in plasma or serum. Because of the short half-life of the biologically active 1,25(OH)D form, this Vitamin D assay primarily detects the more metabolically stable forms, 25(OH)D3 and 25(OH)D2. Accurate detection of these forms requires that they be displaced from the vitamin D binding protein prior to measurement. A simple, acetone-based purification protocol is included as the initial procedure of the Vitamin D assay kit for this purpose.