Quantitative EIA for the determination of 25-hydroxyvitamin D (25-OH D) and other hydroxylated metabolites in human serum or plasma. This method calculates the percentage binding B/B0% of each sample from the zero calibrator (Standard1). A calibration curve is prepared on semi-log axes plotting B/B0% against concentration values with a Four Parameter Logistic curve (4PL) fitted through the data. The the nmol/L is calculated for each sample with specified dilution factor values applied. Supply your absorbance data as reference corrected (i.e. measured at 450nm with reference measured at 650nm).