Devoll R E, Pinero G J, Appelbaum E R, Dul E, Troncoso P, Butler W T, Farach-Carson M C
Department of Basic Sciences, The University of Texas-Houston, Health Science Center, Dental Branch, 6516 John Freeman Avenue, Houston, Texas 77030, USA.
Calcif Tissue Int. 1997 Apr;60(4):380-6. doi: 10.1007/s002239900247.
Studies to assess osteopontin (OPN) localization in adult human bone using immunochemical techniques produce conflicting results due to variations in tissue processing or antibody immunoreactivity. The present study was designed to resolve these discrepancies using well-characterized antibodies and improved antigen detection. An anti-osteopontin (alpha-OPN) antiserum was developed that recognizes various soluble molecular weight forms of human OPN, including monomeric, cleaved, and dimerized products. An affinity column of full length recombinant human OPN (rOPN) coupled to support was used to purify alpha-OPN antibodies. Western analysis showed that the affinity-purified antibodies recognized numerous molecular weight forms of OPN. These antibodies were used to study the distribution of OPN in adult human bone using immunohistochemical techniques combined with an antigen retrieval protocol utilizing a newly developed antigen retrieval solution, Retriev-Alltrade mark (Bronco Technologies Inc, Pasadena, TX). Immunolocalization of OPN in archival bone specimens prior to antigen retrieval produced no demonstrable immunostaining even at high concentrations of alpha-OPN. Use of the antigen retrieval protocol restored OPN immunoreactivity, with strong staining apparent in cement lines, osteoblasts, osteocytes, canaliculi, osteoid, and bone matrix. We conclude that antigen retrieval restores immunochemical recognition of OPN in archival specimens containing bone without increasing nonspecific binding.
利用免疫化学技术评估骨桥蛋白（OPN）在成人骨骼中的定位的研究，由于组织处理或抗体免疫反应性的差异而产生了相互矛盾的结果。本研究旨在使用特性明确的抗体和改进的抗原检测方法来解决这些差异。开发了一种抗骨桥蛋白（α-OPN）抗血清，它能识别多种可溶性分子量形式的人OPN，包括单体、裂解产物和二聚体产物。使用与载体偶联的全长重组人OPN（rOPN）亲和柱来纯化α-OPN抗体。蛋白质印迹分析表明，亲和纯化的抗体识别多种分子量形式的OPN。这些抗体用于通过免疫组织化学技术结合使用新开发的抗原修复溶液Retriev-All（商标，Bronco Technologies Inc，帕萨迪纳，德克萨斯州）的抗原修复方案来研究OPN在成人骨骼中的分布。在抗原修复之前，对存档骨标本中OPN进行免疫定位，即使在高浓度的α-OPN下也没有明显的免疫染色。使用抗原修复方案恢复了OPN的免疫反应性，在黏合线、成骨细胞、骨细胞、骨小管、类骨质和骨基质中可见强烈染色。我们得出结论，抗原修复可恢复含骨存档标本中OPN的免疫化学识别，而不会增加非特异性结合。
