IMMUNOFLUORESCENT LOCALIZATION OF VITAMIN D-DEPENDENT CALCIUM-BINDING PROTEIN

The vitamin D-induced calcium-binding protein (CaBP) present in chick intestinal mucosa was purified and used for the preparation of an antiserum in rabbits. After adsorption the antiserum was judged to be specific for CaBP by immunoelectrophoresis in three different systems. In addition the antiserum was capable of inhibiting the calcium-binding activity of CaBP as measured by equilibrium dialysis. Using the antiserum in micro-Ouchterlony double diffusion tests the previously reported tissue distribution of CaBP in vitamin D-replete chicks was confirmed, i.e., all segments of small intestine and kidney. In addition, the protein was detectable in homogenates of colon from vitamin D-treated chicks, but not in blood plasma, liver, pancreas or bone. The sensitivity of the double diffusion reaction indicated that if CaBP were present in bone it would be at a concentration less than 10 µg/g bone cells (dry weight). Fluoresceinisothiocyanate-conjugated sheep-anti-rabbit γ-globulins were used in the indirect fluorescent antibody localization of CaBP in 4-6-µ cryostat-prepared sections of chick intestinal tissue. Specific fluorescence, indicative of the presence of CaBP, was noted in the periodic acid-Schiff-positive goblet cells and in association with the surface coat-microvillar region of all intestinal epithelial cells in sections from normal and vitamin D-treated rachitic chicks. Immunologically blocked controls and sections from rachitic chicks demonstrated nonspecific fluorescence only.