Identification of osteoclast-specific monoclonal antibodies.

Abstract

Studies on the origin, identification, and characterization of osteoclasts have been difficult. This is in part due to a lack of definitive osteoclast markers and the similarity of these cells in form and function to cells of the mononuclear phagocyte system. To solve this problem, we inoculated isolated chick osteoclasts into mice to generate osteoclast-specific monoclonal antibodies. Supernatants from growth-positive hybridomas were screened by indirect immunofluorescent methods against cultured osteoclasts, monocyte-derived multinucleated giant cells, cultured monocytes, fibroblasts, and limb mesenchyme. Select hybridomas were cloned to produce 375 clones, which were analyzed as described above. Antibody from select clones was also reacted with paraffin sections of bone. In addition, two clones have been analyzed by enzyme-linked immunosorbent asssay (ELISA) and Western blot analysis. Antibody binding from an osteoclast-specific clone and a clone reactive with osteoclasts, giant cells, and cultured monocytes (as determined by immunohistochemical assay) was confirmed by antibody-binding and titration curves quantitated by ELISA. The above studies demonstrate that osteoclast specific antigens exist, and that osteoclasts, giant cells, and cultured monocytes share common determinants not found on other cells screened.The multinucleated osteoclast serves as the major cell type responsible for degradation of bone matrix, and its importance in bone growth, skeletal remodeling, and electrolyte homeostasis has been well established (1-3). The mechanisms and regulation of osteoclast-mediated bone resorption are not yet clearly understood; moreover, little is known about the recruitment and differentiation of osteoclast precursors (4, 5). It is generally believed that osteoclasts originate from cells belonging to the mononuclear phagocyte system (6-8). Although different, macrophages and osteoclasts share certain similarities in form and function, including membrane folding (9), positive acid phosphatase staining, activity of other lysosomal enzymes, and the ability to degrade connective tissue (2, 10). Chick-quail chimera studies (l l, 12) have demonstrated a blood-borne origin for the osteoclast, and parabiosis and marrow transplantation have been successfully used to correct the osteoclast defect in osteopetrosis (13,14). Together, these observations support the hypothesis that osteoclasts are polykaryons that derive from circulating mononuclear cells belonging to the monocyte-macrophage family.In various cell populations, a seemingly homogeneous phenotype can now be divided into functional subpopulations with monoclonal antibodies raised to unique cell surface components. In this regard, mononuclear phagocytes have been shown to play important roles as antigen-presenting cells (15), cytotoxic effector cells (16), and also as secretory cells 1592 (17). Recently, it has been demonstrated by the use of monoclonal antibody technology that one cell type may not be responsible for all functions. In this context, ...

Regulation of Avian Osteoclastic H+-ATPase and Bone Resorption by Tamoxifen and Calmodulin Antagonists

“…The antibody to some extent outlines the cells, suggesting membrane-associated antigen (Fig. 1B, epifluorescence) as reported (18). Similarly, essentially all purified cells are tartrateresistant acid phosphatase-positive (Fig.…”

“…Bone-attached cells were characterized by tartrate-resistant acid phosphatase staining and using a monoclonal antibody, 121F, reacting specifically with avian osteoclasts (Fig. 1), kindly supplied by Philip Osdoby, Washington University, Saint Louis, MO (18). Cells were fixed 2 h in 1% phosphate-buffered formalin at 4°C and incubated 30 min at 20°C with 121F antibody diluted 1:100 or an equivalent dilution of nonimmune ascites in phosphate-buffered saline with 0.05% polyoxyethylene sorbitan monooleate and 1% bovine serum albumin, washed, and incubated with fluoresceinated goat anti-mouse antibody (Sigma) at 1:500 dilution to identify bound primary antibody, washed, and examined by epifluorescence using 450 -490 nm excitation and a 520-nm barrier filter.…”

“…Activity-Because variable findings of steroid effects on bone are reported (3,24), the cell preparations used were affinity-purified and the composition of the resultant cell preparations was characterized using the monoclonal antibody 121F specific for avian osteoclasts (18) and by tartrate-resistant acid phosphatase activity. Essentially all of the material in these preparations stains with the osteoclast-specific antibody ( Fig.…”

Osteoclastic resorption of Ca-P biomaterials implanted in rabbit bone

“…Recently, specific antibodies were raised against some osteoclast cell surface antigens, and immunohistochemical methods were proposed for osteoclast identification [29][30][31][32].…”

A new cytometric method for the immunophenotypic characterization of bone‐derived human osteoclasts

“…Although it is not specific, megakaryocytic lineage cells are TRAP Ϫ . Finally, previous studies have shown that the 121F mAb is specific for osteoclasts (23). Therefore, cells stained with the 121F mAb in bone-derived samples characterized by FCM can be considered to be human osteoclast-like cells due to their morphologic and cytochemical properties.…”