Characteristics of antisera to antigenic forms of 1,25-dihydroxycholecalciferol

Antisera to 1,25-dihydroxycholecalciferol were raised to 1,25-dihydroxycholecalciferol 25-hemisuccinate-bovine serum albumin, 1,25-dihydroxycholecalciferol 3-hemisuccinate-bovine serum albumin, 1,25-dihydroxycholecalciferol 3-hemisuccinate-porcine thyroglobulin and (5Z,7E)-(1S,3R)-1,3 dihydroxy-9,10-seco-24,25, 26,27-tetrakisnor-5,7,10(19)-cholestatrien-23-oic acid porcine thyroglobulin in rabbits. The antisera cross-reacted with a wide spectrum of vitamin D metabolites but their affinity was highest for 1,25-dihydroxycholecalciferol and some differentiated between ergo- and cholecalciferol metabolites. The presence of vitamin D binding protein and the pH of incubation markedly affected the sensitivity and specificity of antisera. A number of antisera were capable of measuring the normal plasma concentrations of vitamin D metabolites and their different affinities could be used for the measurement of 1,25-dihydroxyergocalciferol and 1,25-dihydroxycholecalciferol in plasma. None however were specific enough for the direct measurement of the metabolites in plasma. Immunisation increased the plasma concentration of endogenous 1,25-dihydroxyergo- and cholecalciferol in the animals.