Immunoblot analysis of proteins associated with HEMA-MMA microcapsules: Human serum proteins in vitro and rat proteins following implantation
Immunoblot analysis of proteins associated with HEMA-MMA microcapsules: Human serum proteins in vitro and rat proteins following implantation
复制标题
DOI：
10.1016/s0142-9612(97)00242-1
复制
发表时间：
1998-04-01
期刊：
影响因子：
14
通讯作者：
Sefton, MV
中科院分区：
文献类型：
--
作者：
Babensee, JE;Cornelius, RM;Sefton, MV
使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 和免疫印迹分析对与甲基丙烯酸羟乙酯-甲基丙烯酸甲酯 (HEMA-MMA) 共聚物微胶囊相关的人血清蛋白及其片段进行了表征。将胶囊与血清在体外孵育1周，然后溶解在乙醇中以也沉淀吸附的蛋白质。将沉淀物溶解在 2% (w/v) SDS（“胶囊洗脱液”）中以通过电泳进行测定。免疫印迹中探测到的大多数蛋白质在胶囊洗脱液中被检测到。这些包括纤连蛋白、纤溶酶原、IgG、玻连蛋白、B 因子、H 因子、I 因子、C3，但不包括 β-脂蛋白、纤维蛋白原、HMWK 或 IgM。在胶囊洗脱液和不含胶囊的含血清培养基的免疫印迹中均检测到补体激活片段。因此，独立于胶囊存在而形成的这些片段的吸附可能部分或完全负责与胶囊相关的补体片段。在为期一周的孵育开始时，通过添加 5.8 mM EDTA 来防止补体激活，导致与胶囊相关的低分子量 C3 片段减少。使用抗人抗体植入大鼠腹膜腔1天后，在“自由漂浮”胶囊的洗出液的免疫印迹中也检测到了大鼠蛋白质。检测到的蛋白质包括 HMWK、纤维蛋白原、抗凝血酶 III、转铁蛋白、α(1)-抗胰蛋白酶、纤连蛋白、白蛋白、α(2)-巨球蛋白、玻连蛋白、β(2)-微球蛋白、B 因子和 I 因子。在这些免疫印迹中还检测到大鼠纤维蛋白原、IgG 和补体 C3 片段，但使用的是针对大鼠蛋白的单克隆抗体。 (C) 1998 Elsevier Science Ltd. 保留所有权利。
Human serum proteins and their fragments, associated with hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer microcapsules, were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. Capsules were incubated with serum for 1 week in vitro and then dissolved in ethanol to also precipitate the adsorbed protein. The precipitate was dissolved in 2% (w/v) SDS (the 'capsule eluate') to be assayed by electrophoresis. The majority of proteins probed for in the immunoblots were detected in the capsule eluates. These included fibronectin, plasminogen, IgG, vitronectin, Factor B, Factor H, Factor I, C3, but not beta-lipoprotein, fibrinogen, HMWK, or IgM. Complement activation fragments were detected in both the immunoblots of the capsule eluates and the medium containing serum without capsules. Thus, the adsorption of these fragments, formed independent of capsule presence, may be partially or completely responsible for the complement fragments associated with capsules. The prevention of complement activation by the addition of 5.8 mM EDTA, at the beginning of the week-long incubation, resulted in fewer low-molecular-weight C3 fragments associated with capsules. Rat proteins were also detected in immunoblots of the eluate of 'free-floating' capsules from the rat peritoneal cavity following implantation for 1 day using anti-human antibodies. Detected proteins included HMWK, fibrinogen, antithrombin III, transferrin, alpha(1)-antitrypsin, fibronectin, albumin, alpha(2)-macroglobulin, vitronectin, beta(2)-microglobulin, Factor B and Factor I. Rat fibrinogen, IgG, and complement C3 fragments were also detected in these immunoblots, but with monoclonal antibodies against the rat proteins. (C) 1998 Elsevier Science Ltd. All rights reserved.