Monoclonal antibodies to native noncollagenous bone-specific proteins.

Abstract

Hybridoma technology was used for preparation of murine monoclonal antibodies of high titer against bone-Gla protein and osteonectin. A procedure of immunization and hybridization similar to that already described [Katzmann, J. A., Nesheim, M. E., Hibbard, L. S. & Mann, K. G.(1981) Proc. Nati. Acad. Sci. USA 78,[162][163][164][165][166] and Foster, W. B., Katzmann, J. A., Miller, R. S., Nesheim, M. E. & Mann, K. G. (1982) Thromb. Res. 28,[649][650][651][652][653][654][655][656][657][658][659][660][661] was used. However, in contrast to earlier studies, mice were immunized with an unfractionated protein mixture that had been extracted from bone under nondenaturing conditions. The extract was labeled with 125I by the chloramine-T method. After fusion and initial hybrid growth, screening was accomplished by a solid-phase radioimmunoassay with total 125I-labeled bovine bone protein extract as the tracer. The identities of antibodybound 125I-labeled proteins were assessed by dissolution of the solid-phase immune complex in NaDodSO4 and subsequent electrophoresis and autoradiography. Clones producing specific antibody to a single protein were selected by limiting dilution. The identity of the proteins against which the specific antibodies were produced was confirmed by immunoprecipitation, electrophoresis, and autoradiography. From two fusions, 30 positive hybrids to bone-Gla protein were identified; 7 of these were subcloned and 1 has been expanded as an ascites tumor. One hybrid population was positive for osteonectin, a Mr 15,000 peptide, and for bone-Gla protein. By limiting dilution, the osteonectin clone was selected and subsequently expanded as an ascites tumor. Titration curves made using the respective 1251-labeled purified proteins show the ascites tumors to be producing antibody of high titer (I50 = 10-6) for anti-bone-Gla protein and (I50 -10-5) for antiosteonectin.

Regulation of human bone marrow-derived osteoprogenitor cells by osteogenic growth factors.

“…Immune-adherent bone antigen-positive cells were cultured in serum-free tissue culture medium (9) containing plasminogen-free fibrinogen, which was treated with thrombin (35) to form a fibrin clot. After 7 d of culture, the fibrin clots were dried to a film and subjected to immunocytochemical analysis using monoclonal antibodies to human BGP (osteocalcin) as described elsewhere (10). bone marrow-derived bone precursor cells were not hematogenous in origin, as these cells did not express the pan-hematopoietic cell surface antigen CD34 and failed to respond to hematopoietic growth factors (9).…”

Noncollagenous Bone Matrix Proteins, Calcification, and Thrombosis in Carotid Artery Atherosclerosis

“…54 This role seems to prevent excessive mineralization in bone. Osteonectin and osteocalcin are detectable in normal plasma and serum 40,55 and together with osteopontin are also present in human platelets. 7,27,28 The results on the distribution of these 3 bone-related proteins, osteonectin, osteopontin, and osteocalcin, in carotid artery atherosclerotic plaques have shown that they colocalize with large calcium deposits in clinically significant type V and type VI lesions.…”

“…The MoAbs used were as follows: IIIA 3 A 8 (10 g/mL) to osteonectin and G12 (2 g/mL) to osteocalcin. 40 In normal vessels and placental tissue, osteonectin was detected in ECs, smooth muscle cells (SMCs), and decidua cells as previously described. 11 Osteocalcin was not detected in any cell type.…”

SPARC, a Matricellular Glycoprotein with Important Biological Functions

“…It has therefore been implicated in the mineralization of cartilage and bone. This N-terminal domain contains the major immunological epitopes of SPARC (Stenner et al 1984;Malaval et al 1991). It also exhibits the most divergent sequence among the family of SPARC-like proteins (discussed below).…”