Osteoclast-specific monoclonal antibodies coupled to magnetic beads provide a rapid and efficient method of purifying avian osteoclasts

P. Collin-Osdoby, Merry Jo Oursler, D. Webber, P. Osdoby

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Osteoclasts are the major cell type responsible for normal and pathologic bone resorption. Obtaining highly purified populations of these multinucleated cells has been problematic, although such populations would greatly facilitate investigations of osteoclast regulation and activity. A new immunomagnetic protocol has been devised to surmount these difficulties, employing avian osteoclast-directed monoclonal antibodies (designated 121F, 35L, and 75B) surface coupled to uniformly small, magnetic polystyrene beads covalently conjugated with sheep antimouse IgG. Presentation of these antiosteoclast antibody-coated beads to mixed cell preparations derived from marrow-depleted, collagenase- and/or trypsin-treated chick tibiae and wing bones, followed by magnetic separation and washing, results in efficient and selective binding of osteoclasts to the immunomagnetic beads within minutes. The specific nature of this bead-cell interaction is further demonstrated by the progressive decline in antiosteoclast antibody-coated bead binding to osteoclasts pretreated with the soluble antiosteoclast antibody and also by the absence of binding to osteoclasts by uncoated beads or beads coated with an irrelevant antibody. Under optimal conditions, these isolations typically yield more than a 100-fold enrichment and greater than a 90% purification of osteoclasts from subpopulations of either predominantly nonviable or viable osteoclasts. Although scanning electron microscopy reveals that immunomagnetically purified and cultured osteoclasts internalize large numbers of the antibody-coated beads, such cells appear unimpaired in their ability to attach to tissue culture plastic or devitalized cortical bone slices and to produce resorption pits characteristic for osteoclasts. Additional studies to ascertain the most effective method for removal (desorption) of antibody-coated beads from magnetically isolated osteoclasts demonstrate that moderate physical agitation is at present the most effective protocol to dislodge antibody-coated beads from the cell surface while maintaining osteoclast viability and function. This immunomagnetic technique therefore provides a gentle method for the isolation of highly purified poplations of osteoclasts from heterogeneous bone cell populations in a rapid, efficient, and selective manner.

Original languageEnglish (US)
Pages (from-to)1353-1365
Number of pages13
JournalJournal of Bone and Mineral Research
Volume6
Issue number12
StatePublished - 1991
Externally publishedYes

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Osteoclasts
Monoclonal Antibodies
Antibodies
Population
Bone and Bones
Polystyrenes
Collagenases
Bone Resorption
Tibia
Cell Communication
Electron Scanning Microscopy
Trypsin
Plastics
Sheep
Immunoglobulin G
Bone Marrow

ASJC Scopus subject areas

  • Surgery

Cite this

Osteoclast-specific monoclonal antibodies coupled to magnetic beads provide a rapid and efficient method of purifying avian osteoclasts. / Collin-Osdoby, P.; Oursler, Merry Jo; Webber, D.; Osdoby, P.

In: Journal of Bone and Mineral Research, Vol. 6, No. 12, 1991, p. 1353-1365.

Research output: Contribution to journalArticle

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abstract = "Osteoclasts are the major cell type responsible for normal and pathologic bone resorption. Obtaining highly purified populations of these multinucleated cells has been problematic, although such populations would greatly facilitate investigations of osteoclast regulation and activity. A new immunomagnetic protocol has been devised to surmount these difficulties, employing avian osteoclast-directed monoclonal antibodies (designated 121F, 35L, and 75B) surface coupled to uniformly small, magnetic polystyrene beads covalently conjugated with sheep antimouse IgG. Presentation of these antiosteoclast antibody-coated beads to mixed cell preparations derived from marrow-depleted, collagenase- and/or trypsin-treated chick tibiae and wing bones, followed by magnetic separation and washing, results in efficient and selective binding of osteoclasts to the immunomagnetic beads within minutes. The specific nature of this bead-cell interaction is further demonstrated by the progressive decline in antiosteoclast antibody-coated bead binding to osteoclasts pretreated with the soluble antiosteoclast antibody and also by the absence of binding to osteoclasts by uncoated beads or beads coated with an irrelevant antibody. Under optimal conditions, these isolations typically yield more than a 100-fold enrichment and greater than a 90{\%} purification of osteoclasts from subpopulations of either predominantly nonviable or viable osteoclasts. Although scanning electron microscopy reveals that immunomagnetically purified and cultured osteoclasts internalize large numbers of the antibody-coated beads, such cells appear unimpaired in their ability to attach to tissue culture plastic or devitalized cortical bone slices and to produce resorption pits characteristic for osteoclasts. Additional studies to ascertain the most effective method for removal (desorption) of antibody-coated beads from magnetically isolated osteoclasts demonstrate that moderate physical agitation is at present the most effective protocol to dislodge antibody-coated beads from the cell surface while maintaining osteoclast viability and function. This immunomagnetic technique therefore provides a gentle method for the isolation of highly purified poplations of osteoclasts from heterogeneous bone cell populations in a rapid, efficient, and selective manner.",
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