Bertozzi C R, Singer M S, Rosen S D
Department of Anatomy, University of California, San Francisco 94143-0452, USA.
J Immunol Methods. 1997 Apr 25;203(2):157-65. doi: 10.1016/s0022-1759(97)00026-4.
Members of the selectin family of adhesion receptors, consisting of L-, P- and E-selectin, mediate the initial interaction between leukocytes and endothelium during leukocyte trafficking from the blood into tissue sites. These receptors have attracted great attention in recent years due to their participation in a number of acute and chronic inflammatory diseases. We describe here a new ELISA that measures the binding between selectin-IgG chimeras and a physiological ligand for L-selectin and can be used to screen selectin inhibitors. The ligand used is a mucin-like glycoprotein known as GlyCAM-1, which is derived from high endothelial venules in secondary lymphoid organs. We demonstrate binding of all three selectins to GlyCAM-1 and demonstrate that the binding interactions satisfy a number of important criteria. The advantage of this ELISA over previous assays is that a macromolecular physiological ligand is employed, rather than a fortuitous or simplified carbohydrate ligand. Thus, the protein-carbohydrate interactions, as well as other interactions contributing to ligand recognition, can be investigated. The assay is suitable for high-throughout screening of compounds and may find use in the identification of selectin antagonists with anti-inflammatory potential.
选择素家族黏附受体成员包括L-选择素、P-选择素和E-选择素，在白细胞从血液进入组织部位的过程中，介导白细胞与内皮细胞之间的初始相互作用。近年来，由于这些受体参与了多种急慢性炎症性疾病，因此备受关注。我们在此描述一种新的酶联免疫吸附测定法（ELISA），该方法可检测选择素-IgG嵌合体与L-选择素的生理配体之间的结合，可用于筛选选择素抑制剂。所使用的配体是一种称为GlyCAM-1的黏蛋白样糖蛋白，它源自次级淋巴器官中的高内皮微静脉。我们证明了所有三种选择素与GlyCAM-1的结合，并证明这种结合相互作用满足许多重要标准。与以前的检测方法相比，这种ELISA的优点是使用了大分子生理配体，而不是偶然的或简化的碳水化合物配体。因此，可以研究蛋白质-碳水化合物相互作用以及其他有助于配体识别的相互作用。该检测方法适用于化合物的高通量筛选，并可能用于鉴定具有抗炎潜力的选择素拮抗剂。
