Characterization of human 1,25-dihydroxyvitamin D 3 receptor anti-peptide antibodies

Abstract

Rabbit and chicken antibodies were raised against two peptides synthesized according to the structure of human 1,25-dihydroxyvitamin D3 receptor (hVDR): rabbit αhVDR-103 against the N-terminal amino acids 5–18 and αhVDR-104 against the amino acids 172–186 in the hinge region and chicken αhVDR-cab11 against the amino acids 172–186, respectively. The specificity of the antibodies was tested by peptide saturation, SDS-PAGE immunoblotting, gel shift assay and sucrose gradient centrifugation. Immunoblotting of a soluble extract (cytosol) from osteosarcoma cell line MG-63 showed a single band with an M r of about 48,000 and human intestine cytosol a broad band (50–63,000) for both antibodies. The antibodies recognized activated (3.2S) hVDR by shifting the centrifugation sedimentation profile to 5–6S. The antibodies showed nuclear immunostaining of unoccupied VDR in human osteosarcoma cells MG-63, U2-Os and SaOs-2. The immunoreaction could be satrated with the coresponding synthetic peptide. In immunoblot αhVDR-103 reacted with human and rat VDR, whereas αhVDR-104 recognized human VDR only. Similarly in immunohistochemistry, αhVDR-103 showed staining with hVDR and rVDR, whereas αhVDR-104 reacted only with hVDR. All antibodies recognized the native hVDR as verified with sucrose gradient centrifugation or immunoprecipitation but only αhVDR-103 and αhVDR-cab11 in gel shift assay of hVDR associated with the vitamin D-responsive element of human osteocalcin gene promoter.