Vitamin D-Dependent Rat Renal Calcium-Binding Protein: Development of a Radioimmu no assay, Tissue Distribution, and Immunologic Identification*

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Abstract
A sensitive double antibody radioimmunoassay [RIA] has been developed for the 28,000 MW rat renal vitamin D-dependent Ca-binding protein. Using this assay, concentrations of Ca-binding protein (CaBP) as low as 30 ng can be measured. The assay is precise (intraasay variability, 5.0%) and reproducible (interassay variability, 8.2%). Measurements of renal CaBP by RIA showed a good correlation with measurements of CaBP by the chelex resin assay and by polyacrylamide gel analysis by densitometric tracing using a purified CabP marker. The concentration of CaBP in the vitamin D-replete rat kidney is 7.3 .+-. 1.0 (mean .+-. standard error of the mean) .mu.g/mg protein. In vitamin D-deficient rats the level of renal CaBP is 2.6 .+-. 0.3 .mu.g/mg protein. Tissue distribution of immunoreactive rat renal CaBP showed the highest concentration of CaBP in the rat cerebellum (38.3 .+-. 5.1 .mu.g/mg protein). Lower concentrations of immunoreactive CaBP were detected in several other rat tissues. No immunoreactive CaBP was detected in rat or human serum. In necropsy human kidney and cerebellum, high levels of immunoreactive CaBP were also detected (1.5 .+-. 0.1 and 27.3 .+-. 2.1 .mu.g/mg protein, respectively). When extracts of rat kidney, brain, human cerebellum and kidney were assayed at several dilutions, immunodisplacement curves parallel to that of pure renal CaBP were observed, indicating immunochemical similarity. Fractionation of extracts of rat cerebellum, human kidney and human cerebellum on Sephadex G-100 recreated immunoreactivity and Ca-binding activity in the 28,000 MW region similar to rat kidney.