Keski-Oja J, Lyons R M, Moses H L
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Cancer Res. 1987 Dec 15;47(24 Pt 1):6451-8.
In an attempt to identify and quantitate latent and active forms of transforming growth factor beta (TGF beta) without the use of cell cultures and to test for autocrine stimulation by TGF beta, rabbit antibodies were raised against native human and porcine platelet-derived TGF beta. A radioimmunoassay for TGF beta was developed using radioiodinated TGF beta, anti-TGF beta antibodies, and protein A. Inhibition in the radioimmunoassay was achieved with nanogram quantities of TGF beta, comparable to the sensitivity of radioreceptor assays. Analyses of the TGF beta levels of conditioned medium from cultured cells indicated that the latent form(s) of TGF beta is not detectable in the radioimmunoassay established using antibodies raised against native TGF beta. Immunoprecipitation analysis of radiolabeled conditioned medium revealed a specific Mr 25,000 band only after acidification. A Mr 62,000 protein was observed with and without prior acidification of the medium but could not be competed with unlabeled TGF beta in the immunoprecipitation indicating antigenic unrelatedness. The anti-TGF beta IgG inhibited the binding of [125I]TGF beta to the cell surface receptors in a radioreceptor assay. TGF beta inhibition of A549 cell growth was reversed by the antibodies, which also neutralized the growth inhibitory effects of TGF beta on AKR-2B cells in a monolayer [3H]thymidine incorporation assay as demonstrated by prevention of TGF beta inhibition of insulin and epidermal growth factor-stimulated DNA synthesis. The antibodies also effectively inhibited spontaneous soft agar growth of AKR-MCA fibroblasts, providing evidence for autocrine secretion of TGF beta as a mechanism of their anchorage-independent growth.
为了在不使用细胞培养的情况下鉴定和定量转化生长因子β（TGF-β）的潜伏形式和活性形式，并检测TGF-β的自分泌刺激作用，制备了针对天然人及猪血小板衍生TGF-β的兔抗体。利用放射性碘化TGF-β、抗TGF-β抗体和蛋白A开发了一种TGF-β放射免疫测定法。该放射免疫测定法对纳克量的TGF-β有抑制作用，与放射受体测定法的灵敏度相当。对培养细胞条件培养基中TGF-β水平的分析表明，在用针对天然TGF-β产生的抗体建立的放射免疫测定法中检测不到TGF-β的潜伏形式。对放射性标记的条件培养基进行免疫沉淀分析，仅在酸化后才显示出一条特定的分子量为25000的条带。无论培养基是否预先酸化，都观察到一条分子量为62000的蛋白条带，但在免疫沉淀中不能被未标记的TGF-β竞争，表明其抗原性不相关。在放射受体测定中，抗TGF-β IgG抑制了[125I]TGF-β与细胞表面受体的结合。抗体逆转了TGF-β对A549细胞生长的抑制作用，在单层[3H]胸苷掺入试验中，抗体还中和了TGF-β对AKR-2B细胞生长的抑制作用，这通过防止TGF-β抑制胰岛素和表皮生长因子刺激的DNA合成得到证明。这些抗体还有效抑制了AKR-MCA成纤维细胞在软琼脂中的自发生长，为TGF-β的自分泌作为其不依赖贴壁生长的机制提供了证据。
