Characterization of human 1,25-dihydroxyvitamin D3 receptor anti-peptide antibodies

• Published in: The Journal of steroid biochemistry and molecular biology Vol. 43; no. 7; pp. 649 - 657
• Main Authors: TUOHIMAA, P, BLÄUER, M, JÄÄSKELÄINEN, T, ITKONEN, A, LINDFORS, M, MAHONEN, A, PALVIMO, J, VILJA, P, MÄENPÄÄ, P. H
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Science 01.12.1992
• Subjects: Amino Acid Sequence; Animals; Antibodies - chemistry; Antibody Specificity; Antigen-antibody reactions, antigen-antibody complexes, antibody-complement and others. Study of affinity. Antigen presentation; Biological and medical sciences; Centrifugation, Density Gradient; Chickens; Female; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Immunoblotting; Immunohistochemistry; Male; Molecular immunology; Molecular Sequence Data; Osteosarcoma - immunology; Peptides - immunology; Precipitin Tests; Rabbits; Receptors, Calcitriol; Receptors, Steroid - immunology; Tumor Cells, Cultured; Characterization; Human; Immunoglobulins; Vitamin D; Steroid hormone; Antibody; Cholecalciferol(1,25-dihydroxy); Hormonal receptor
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/0960-0760(92)90290-Y

Rabbit and chicken antibodies were raised against two peptides synthesized according to the structure of human 1,25-dihydroxyvitamin D3 receptor (hVDR): rabbit alpha hVDR-103 against the N-terminal amino acids 5-18 and alpha hVDR-104 against the amino acids 172-186 in the hinge region and chicken alpha hVDR-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 saturated with the corresponding synthetic peptide. In immunoblot alpha hVDR-103 reacted with human and rat VDR, whereas alpha hVDR-104 recognized human VDR only. Similarly in immunohistochemistry, alpha hVDR-103 showed staining with hVDR and rVDR, whereas alpha hVDR-104 reacted only with hVDR. All antibodies recognized the native hVDR as verified with sucrose gradient centrifugation or immunoprecipitation but only alpha hVDR-103 and alpha hVDR-cab11 in gel shift assay of hVDR associated with the vitamin D-responsive element of human osteocalcin gene promoter.