LUMINESCENCE ASSAY FOR PHARMECEUTICAL SCREENING

用于药物筛选的发光测定

• 批准号: 3767487
• 负责人: A EIDSATH
• 金额: --
• 依托单位: NATIONAL CENTER FOR RESEARCH RESOURCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3767487
• 关键词: Xenopus oocyte; biomedical automation; biomedical equipment development; calcium indicator; calcium metabolism; drug interactions; human tissue; luminescence

The gene for the photoprotein aequorin has been incorporated into several mammalian cell lines. Because the luminescence of this protein reflects the local Ca++ concentration, it is very useful for assaying cellular Ca++ signals that are activated, or blocked, by drugs of potential therapeutic value. A major experimental difficulty lies in the very low level of light that is emitted, since only one photon is liberated by each molecule of aequorin, and we have calculated that there are only 20,000 molecules per cell. We plan to modify a photon-counting instrument that we built to measure luminescence from frog oocytes. The photon count will be used as an assay to determine the activity of drugs that influence cell Ca++ metaboism. Large numbers of chemicals will be screened for activity, so the instrument will be automated as much as possible. We have the difficult job of injecting small quantities (20 to 50 microliters) of fluids and cell suspensions. If the preliminary results are positive, we will consider purchasing an autosampler to go along with the instrument. Another of the difficulties in the design is the ~sticky~ character of many of the drugs, even to stainless steel. Since even small residues from prior experiments could affect the luminescence, all of the reusable pieces and tubing will have to be made of Teflon~.

发光蛋白水母发光蛋白的基因已被整合到几个基因中 哺乳动物细胞系。 因为这种蛋白质的发光反映了 局部Ca++浓度，这对于测定细胞内Ca ++浓度是非常有用的。 Ca++信号被潜在的药物激活或阻断 治疗价值 一个主要的实验困难在于， 发射的光的水平，因为只有一个光子被释放， 每一个水母发光蛋白分子，我们计算出， 每个细胞有两万个分子 我们计划修改一个光子计数器 这是我们用来测量青蛙卵母细胞发光的仪器。 的 光子计数将用作测定药物活性的分析 影响细胞Ca++代谢。 大量的化学物质将被 筛选活性，因此仪器将尽可能自动化， 可能 我们有困难的工作，注入少量（20 至50微升）的流体和细胞悬浮液。如果初步 结果是积极的，我们将考虑购买自动采样器去 沿着仪器。 设计中的另一个困难是 许多药物的粘性，甚至不锈钢。 因为即使是以前实验中的小残留物也会影响 发光，所有的可重复使用的零件和管道将不得不作出 的Teflon~。