Odeyale C O, Kang Y H
Naval Medical Research Institute, Bethesda, Maryland 20814-5055.
J Histochem Cytochem. 1988 Sep;36(9):1131-7. doi: 10.1177/36.9.3136207.
We describe a procedure for lipopolysaccharide (LPS) biotinylation using N-biotinyl-L-lysine and application of the biotinylated LPS (Bi-LPS) to localization of LPS binding sites and subcellular distribution. Biotinylation of LPS was confirmed by enzyme-linked immunosorbent assay (ELISA), gel immunodiffusion, and immunodot techniques. The biological and toxicological activity of the Bi-LPS was tested by Limulus amoebocyte lysate (LAL) assays and histopathological examinations, respectively. Results showed that biotin was conjugated to LPS without disrupting the biological/toxicological activity of the molecule, which indicates that the biotin is directly linked to the polysaccharide portion of LPS. Localization of binding sites and subcellular distribution of Bi-LPS in human platelets and monocytes were studied by electron microscopy using an avidin-biotin-horseradish peroxidase (HRP) or streptavidin-gold method. Platelet surfaces were intensely stained by the reaction product of horseradish peroxidase (HPR) 5 min after incubation, and Bi-LPS was localized in small vesicles and vacuoles of platelets and in the phagocytic vacuoles of monocytes 60 min post incubation. Bi-LPS provides a reliable, stable, and sensitive tool for determination of LPS binding sites and subcellular distribution.
我们描述了一种使用N-生物素-L-赖氨酸对脂多糖（LPS）进行生物素化的方法，以及将生物素化的LPS（Bi-LPS）应用于LPS结合位点的定位和亚细胞分布研究。通过酶联免疫吸附测定（ELISA）、凝胶免疫扩散和免疫斑点技术确认了LPS的生物素化。分别通过鲎试剂法（LAL）测定和组织病理学检查测试了Bi-LPS的生物学和毒理学活性。结果表明，生物素与LPS结合而不破坏该分子的生物学/毒理学活性，这表明生物素直接连接到LPS的多糖部分。使用抗生物素蛋白-生物素-辣根过氧化物酶（HRP）或链霉抗生物素蛋白-金方法，通过电子显微镜研究了Bi-LPS在人血小板和单核细胞中的结合位点定位和亚细胞分布。孵育5分钟后，辣根过氧化物酶（HPR）的反应产物对血小板表面进行了强烈染色，孵育60分钟后，Bi-LPS定位于血小板的小泡和液泡以及单核细胞的吞噬泡中。Bi-LPS为确定LPS结合位点和亚细胞分布提供了一种可靠、稳定且灵敏的工具。
