Barcellos-Hoff M H, Ehrhart E J, Kalia M, Jirtle R, Flanders K, Tsang M L
Life Sciences Division, Lawrence Berkely Laboratory, University of California, Berkeley 94720, USA.
Am J Pathol. 1995 Nov;147(5):1228-37.
The biological activity of transforming growth factor-beta 1 (TGF-beta) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-beta could provide insight into TGF-beta activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-beta status is known. To provide in situ analysis of antibodies to differentiate between these functional forms, we used xenografts of human tumor cells modified by transfection to overexpress latent TGF-beta or constitutively active TGF-beta. This comparison revealed that, whereas most antibodies did not differentiate between TGF-beta activation status, the immunoreactivity of some antibodies was activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-beta, LC(1-30) and CC(1-30) showed marked preferential immunoreactivity with active TGF-beta versus latent TGF-beta in cryosections. However, in formalin-fixed, paraffin-embedded tissue, discrimination of active TGF-beta by CC(1-30) was lost and immunoreactivity was distinctly extracellular, as previously reported for this antibody. Similar processing-dependent extracellular localization was found with a neutralizing antibody raised to recombinant TGF-beta. Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-beta only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-beta in both frozen and paraffin-embedded tissue. Thus, in situ discrimination of active versus latent TGF-beta depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with specific functional forms of a protein.
转化生长因子-β1（TGF-β）的生物活性受其从潜伏复合物中解离的调控。对活性和潜伏性TGF-β进行免疫组织化学鉴别有助于深入了解生理和病理过程中TGF-β的激活情况。然而，由于缺乏已知TGF-β状态的组织，原位免疫反应特异性的评估受到了阻碍。为了对抗体进行原位分析以区分这些功能形式，我们使用了通过转染修饰以过表达潜伏性TGF-β或组成型活性TGF-β的人肿瘤细胞异种移植物。这种比较表明，虽然大多数抗体无法区分TGF-β的激活状态，但某些抗体的免疫反应性取决于激活状态。两种广泛使用的针对TGF-β氨基末端的肽抗体，LC(1 - 30)和CC(1 - 30)，在冰冻切片中对活性TGF-β与潜伏性TGF-β显示出明显的优先免疫反应性。然而，在福尔马林固定、石蜡包埋的组织中，CC(1 - 30)对活性TGF-β的鉴别能力丧失，免疫反应性明显位于细胞外，正如先前对该抗体的报道。用针对重组TGF-β产生的中和抗体也发现了类似的加工依赖性细胞外定位。抗原修复恢复了两种抗体与细胞相关的免疫反应性。两种针对肽段78 - 109的抗体仅在石蜡切片中对活性TGF-β显示出轻度至中度的优先免疫反应性。LC(1 - 30)是唯一一种在冰冻和石蜡包埋组织中都能区分活性与潜伏性TGF-β的测试抗体。因此，活性与潜伏性TGF-β的原位鉴别取决于抗体和组织制备方法。我们提出，经过工程改造以表达特定蛋白质特定形式的组织提供了一种生理环境，可用于评估抗体与蛋白质特定功能形式的反应性。
