The 25-Hydroxy Vitamin DS EIA assay is an enzyme immunoassay assay. 25 μL of each calibrators, controls and patient sample is diluted with biotin labelled 25(OH)D. The diluted samples are incubated in microtiter wells which are coated with a highly specific sheep 25(OH)D antibody at room temperature before aspiration and washing. Enzyme (horseradish peroxidase) labelled avidin is added and binds selectively to complexed biotin and, following a further wash step, color is developed using a chromogenic substrate (TMB). The absorbance of the stopped reaction mixtures are read in a microtiter plate reader, color intensity developed being inversely proportional to the concentration of 25(OH)D.