George S E, Schaeffer M T, Cully D, Beer M S, McAllister G
Merck, Sharp & Dohme, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR, United Kingdom.
Anal Biochem. 2000 Nov 15;286(2):231-7. doi: 10.1006/abio.2000.4821.
A glow-type aequorin luminescence assay for measuring receptor-mediated stimulation of intracellular calcium levels is described and characterized. The human 5-hydroxytryptamine(2A) receptor stably coexpressed in human embryonic kidney cells with apoaequorin was used to characterize the system and showed that following the flash reaction, a stable luminescence signal could be measured using a microplate scintillation counter for between 3 and 7 h after the addition of receptor agonist. Furthermore, this luminescence was dependent on the concentration of agonist used and gave potency values that were stable over this time period. Testing a range of 5-hydroxytryptamine(2A) receptor agonists gave the expected rank order of potency for this receptor. The glow luminescence could also be inhibited by 5-hydroxytryptamine(2A) receptor antagonists, generating affinity values that directly correlated with those determined for inhibition of the flash reaction carried out under the same buffer conditions. The assay therefore gave pharmacologically relevant data and allows a significant improvement of throughput over the traditional flash-type measurements made using an injecting luminometer.
本文描述并表征了一种用于测量受体介导的细胞内钙水平刺激的辉光型水母发光蛋白发光测定法。将人胚胎肾细胞中与人脱辅基水母发光蛋白稳定共表达的人5-羟色胺（2A）受体用于表征该系统，结果表明，在闪光反应后，加入受体激动剂后3至7小时内，使用微孔板闪烁计数器可测量到稳定的发光信号。此外，这种发光依赖于所用激动剂的浓度，并给出了在该时间段内稳定的效价。测试一系列5-羟色胺（2A）受体激动剂，得到了该受体预期的效价顺序。辉光发光也可被5-羟色胺（2A）受体拮抗剂抑制，产生的亲和力值与在相同缓冲条件下进行的闪光反应抑制测定所确定的值直接相关。因此，该测定法给出了药理学相关数据，并且与使用注射式发光计进行的传统闪光型测量相比，显著提高了通量。
