Okuzaki Daisuke, Kimura Shoichi, Yabuta Norikazu, Ohmine Toshinari, Nojima Hiroshi
Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
BMC Clin Pathol. 2011 Aug 17;11:9. doi: 10.1186/1472-6890-11-9.
Whole-protein extracts from peripheral blood leukocytes are ideal for basic and clinical research. However, lack of a simple preparation technique has limited the use of such extracts. The aim of this study is to develop a simple and easy system that can selectively obtain leukocyte extracts without hemoglobin.
A filter that captures the leukocytes but not RBCs was set at the bottom of a 10-mL medical syringe by sandwiching it between plastic stoppers. The capturing efficiency of leukocytes with this tool, called LeukoCatch, was examined using human macrophage cells (MONO-MAC-6). The abilities of LeukoCatch system to capture the leukocyte proteins and to remove the hemoglobin from RBCs were tested by western blot analysis using human blood samples.
This study presents the development of LeukoCatch, a novel tool that allows the preparation of leukocyte extracts from blood samples within 3 min without centrifugation. Tissue-cultured human macrophage cells were tested to determine the optimal filter numbers and pass-through frequencies of LeukoCatch, which was then applied to 2-mL blood samples. Samples were passed 2~5 times through a LeukoCatch equipped with 5 filters, washed twice with phosphate-buffered saline for red cell removal, and leukocyte proteins were extracted with 0.5 mL of elution buffer. Western blot analysis of the purified extract indicated that more than 90% of hemoglobin was removed by the LeukoCatch and that the protein recovery rate of leukocytes was at least 4 times better than that of the conventional centrifugation method.
We conclude that LeukoCatch is useful not only for diagnosis at the bedside but also for basic research using blood samples or tissue culture cells.
外周血白细胞的全蛋白提取物是基础研究和临床研究的理想材料。然而，缺乏简单的制备技术限制了此类提取物的应用。本研究的目的是开发一种简单易行的系统，能够选择性地获得不含血红蛋白的白细胞提取物。
通过将一种能捕获白细胞而不捕获红细胞的滤器夹在塑料塞之间，置于10毫升医用注射器底部。使用人巨噬细胞（MONO-MAC-6）检测这种名为LeukoCatch的工具捕获白细胞的效率。使用人血样本，通过蛋白质免疫印迹分析测试LeukoCatch系统捕获白细胞蛋白和去除红细胞中血红蛋白的能力。
本研究展示了LeukoCatch的开发，这是一种新型工具，可在无需离心的情况下在3分钟内从血样中制备白细胞提取物。通过检测组织培养的人巨噬细胞来确定LeukoCatch的最佳滤器数量和通过频率，然后将其应用于2毫升血样。样本通过装有5个滤器的LeukoCatch 2至5次，用磷酸盐缓冲盐水洗涤两次以去除红细胞，并用0.5毫升洗脱缓冲液提取白细胞蛋白。对纯化提取物的蛋白质免疫印迹分析表明，LeukoCatch去除了超过90%的血红蛋白，白细胞的蛋白质回收率至少比传统离心法高4倍。
我们得出结论，LeukoCatch不仅有助于床边诊断，还可用于使用血样或组织培养细胞的基础研究。
