Methods include determining an amount of vitamin D analyte in a sample suspected of containing the vitamin D analyte. A combination is provided in an assay medium that includes the sample, a vitamin D epimer antibody that is specific for epimers of the vitamin D analyte wherein the vitamin D epimer antibody does not bind to any detectable degree to the vitamin D analyte, and a vitamin D antibody that is specific for the vitamin D analyte, and. The assay medium is incubated under conditions for binding of the vitamin D epimer antibody to the epimers of the vitamin D analyte and for binding of the vitamin D antibody to the vitamin D analyte to form a vitamin D antibody-bound complex. The amount of vitamin D antibody-bound complex is determined and related to the amount of vitamin D analyte in the sample.
