Aniagolu J, Swartz G M, Dijkstra J, Madsen J W, Raney J J, Green S J
EntreMed, Inc., Rockville, MD 20850, USA.
J Immunol Methods. 1995 May 11;182(1):85-92. doi: 10.1016/0022-1759(95)00026-7.
An analytical immunoblotting procedure and a serological enzyme-linked immunosorbent assay (ELISA) for the characterization of antibodies to cholesterol are described. Hydrophobic membranes consisting of polyvinylidene fluoride (PVDF) are used to immobilize cholesterol for immunodetection by anti-sterol antibodies. To determine whether antibodies to cholesterol were induced after immunization with liposomal cholesterol, we separated total lipid extracts of very-low density lipoproteins by thin layer chromatography (TLC) on silica gel plates and transferred the separated lipid classes to PVDF membranes using isopropanol to facilitate passive diffusion. Lipid transfer was confirmed by exposure of membranes to iodine vapors or by staining of cholesterol with filipin complex. Serum from immunized mice reacted with cholesterol, whereas pre-immune serum or serum from mice injected with control liposomes did not bind. To determine the amount of anti-cholesterol activity in serum, we coated microtiter plates consisting of PVDF membrane wells with cholesterol. The PVDF membrane-based ELISA was found to be more reproducible and four-fold more sensitive than the conventional ELISA on polystyrene plates. These techniques may be useful in the analysis of anti-sterol antibodies and antibodies to other hydrophobic antigens.
本文描述了一种用于鉴定胆固醇抗体的分析免疫印迹法和血清学酶联免疫吸附测定（ELISA）。由聚偏二氟乙烯（PVDF）组成的疏水膜用于固定胆固醇，以通过抗固醇抗体进行免疫检测。为了确定用脂质体胆固醇免疫后是否诱导产生了胆固醇抗体，我们在硅胶板上通过薄层色谱（TLC）分离极低密度脂蛋白的总脂质提取物，并使用异丙醇促进被动扩散，将分离的脂质类别转移到PVDF膜上。通过将膜暴露于碘蒸气或用菲律宾菌素复合物对胆固醇进行染色来确认脂质转移。免疫小鼠的血清与胆固醇反应，而免疫前血清或注射对照脂质体的小鼠血清不结合。为了确定血清中抗胆固醇活性的量，我们用胆固醇包被由PVDF膜孔组成的微量滴定板。发现基于PVDF膜的ELISA比聚苯乙烯板上的传统ELISA更具可重复性，灵敏度高四倍。这些技术可能有助于分析抗固醇抗体和针对其他疏水抗原的抗体。
