Osteoclast-specific monoclonal antibodies coupled to magnetic beads provide a rapid and efficient method of purifying avian osteoclasts.
Osteoclast-specific monoclonal antibodies coupled to magnetic beads provide a rapid and efficient method of purifying avian osteoclasts.
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与磁珠偶联的破骨细胞特异性单克隆抗体提供了一种快速有效的纯化禽类破骨细胞的方法。
DOI：
10.1002/jbmr.5650061213
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Osdoby,P
中科院分区：
文献类型：
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作者：
Collin-Osdoby,P;Oursler,MJ;Webber,D;Osdoby,P
破骨细胞是负责正常和病理性骨吸收的主要细胞类型。获得这些多核细胞的高度纯化的群体一直是有问题的，尽管这样的群体将大大促进破骨细胞调节和活性的研究。已经设计了一种新的免疫磁性方案来克服这些困难，采用禽类破骨细胞定向单克隆抗体（命名为121 F，35 L和75 B）表面偶联到均匀小的磁性聚苯乙烯珠上，聚苯乙烯珠与羊抗鼠IgG共价偶联。将这些抗破骨细胞抗体包被的珠粒呈递给来自骨髓耗尽的、胶原酶和/或胰蛋白酶处理的鸡胫骨和翼骨的混合细胞制剂，然后进行磁性分离和洗涤，导致破骨细胞在几分钟内与免疫磁珠有效和选择性结合。通过抗破骨细胞抗体包被的微珠与用可溶性抗破骨细胞抗体预处理的破骨细胞结合的逐渐下降，以及未包被的微珠或包被有不相关抗体的微珠与破骨细胞不结合，进一步证明了这种微珠-细胞相互作用的特异性。在最佳条件下，这些分离通常产生超过100倍的富集和超过90%的纯化，从主要是无活力或有活力的破骨细胞亚群中分离出破骨细胞。尽管扫描电子显微镜显示免疫磁性纯化和培养的破骨细胞内化了大量抗体包被的珠粒，但这些细胞附着于组织培养塑料或失活皮质骨切片的能力似乎未受损，并产生破骨细胞特有的吸收陷窝。为确定从磁性分离的破骨细胞中去除（解吸）抗体包被微珠的最有效方法而进行的其他研究表明，适度的物理搅拌是目前从细胞表面去除抗体包被微珠同时保持破骨细胞活力和功能的最有效方案。因此，这种免疫磁性技术提供了一种温和的方法，用于以快速、有效和选择性的方式从异质性骨细胞群中分离高度纯化的破骨细胞群。
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.