The ST AIA-PACK 25-OH Vitamin assay consists of a pretreatment step to release 25-OH Vitamin D from its serum transporter. Pretreated samples are then added to a ST AIA-PACK 25-OH Vitamin D test cup that contain 25-OH vitamin D-specific monoclonal antibody immobilized on magnetic beads to which 25-OH Vitamin D binds. Next, 25-OH vitamin D conjugated to bovine alkaline phosphatase is added to the reaction mixture, which competes with 25-OH vitamin D for binding to the antibody on magnetic beads. Subsequently, the magnetic beads are washed to remove the unbound enzyme labeled 25-OH vitamin D and are then incubated with a fluorogenic substrate, 4-methylumbelliferyl phosphate (4MUP). The amount of enzyme-labeled 25-OH vitamin D that binds to the beads is inversely proportional to the 25-OH vitamin D concentration in the test sample.
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