Shin S H, Lim Y, Lee S E, Yang N W, Rhee J H
Department of Microbiology, Chosun University, Medical School, Kwanju 501-759, South Korea.
J Microbiol Methods. 2001 Feb 1;44(1):89-95. doi: 10.1016/s0167-7012(00)00229-3.
We developed a simple and universal method, by modifying the universal CAS (Chrome azurol S) assay, measuring siderophores in various biological fluids. We named the assay as CAS agar diffusion (CASAD) assay. CAS plate devoid of nutrients was prepared by using Bacto-agar (1.5%, w/v) as a matrix. Holes with 5-mm-diameter were punched on the CAS agar plate. Each hole was added by 35 microl of the test fluids containing Desferal that was twofold serially diluted. After incubating at 37 degrees C or room temperature for 4-8 h, the size of orange haloes formed around the holes was measured. The size of orange haloes correlated well with the concentration of Desferal in all the biological fluids tested in this study. CASAD assay showed consistent results in wide pH range from 5 to 9. Addition of iron to the test fluids containing Desferal decreased the size of orange haloes in a dose-dependent manner, which suggests that the CASAD assay detects only iron non-bound siderophore. These results suggest that CASAD assay would serve as a simple, stable, and highly reproducible test for screening and quantitative siderophore analysis in biological fluids.
我们通过改进通用的铬天青S（CAS）测定法，开发了一种简单通用的方法来测量各种生物体液中的铁载体。我们将该测定法命名为CAS琼脂扩散（CASAD）测定法。使用Bacto-琼脂（1.5%，w/v）作为基质制备不含营养物质的CAS平板。在CAS琼脂平板上打孔，孔径为5毫米。每个孔中加入35微升含有去铁胺且经两倍系列稀释的测试液。在37℃或室温下孵育4 - 8小时后，测量孔周围形成的橙色晕圈的大小。在本研究中测试的所有生物体液中，橙色晕圈的大小与去铁胺的浓度密切相关。CASAD测定法在5至9的宽pH范围内显示出一致的结果。向含有去铁胺的测试液中添加铁会以剂量依赖的方式减小橙色晕圈的大小，这表明CASAD测定法仅检测未结合铁的铁载体。这些结果表明，CASAD测定法可作为一种简单、稳定且高度可重复的测试方法，用于生物体液中铁载体的筛选和定量分析。
