Blomberg K, Ulfstedt A C
Wallac Biochemical Laboratory, Abo Akademi University, Turku, Finland.
J Immunol Methods. 1993 Mar 15;160(1):27-34. doi: 10.1016/0022-1759(93)90005-r.
A time-resolved fluorometric assay for the detection of natural killer cell activity against target cells labelled with the fluorescent chelate europium-6,6"-bis[N,N-bis(carboxymethyl)-aminomethyl]-4'-phenyl-2,2',6', 2"-terpyridine (EuCAPT) has been developed. In the assay released EuCAPT from lysed K-562 cells is measured in the supernatant after co-incubation of the target cells with effector cells. Thus, the performance of the assay is essentially similar to the previously described EuDTPA assay and the widely used 51Cr assay. EuCAPT is released from target cells lysed by effector cells faster than 51CrO4(2-) but somewhat slower than EuDTPA. In contrast to methods based on prompt fluorometry the autofluorescence from culture medium supplemented with serum can be avoided by the use of time-resolved fluorometry. The result shows that fluorescent europium chelates provide an alternative to radioactive markers currently used for the assessment of in vitro cellular cytotoxicity.
已开发出一种时间分辨荧光测定法，用于检测自然杀伤细胞对用荧光螯合物铕-6,6”-双[N,N-双(羧甲基)-氨甲基]-4'-苯基-2,2',6',2”-三联吡啶（EuCAPT）标记的靶细胞的活性。在该测定法中，将靶细胞与效应细胞共同孵育后，测量上清液中从裂解的K-562细胞释放的EuCAPT。因此，该测定法的性能与先前描述的EuDTPA测定法和广泛使用的51Cr测定法基本相似。EuCAPT从被效应细胞裂解的靶细胞中释放的速度比51CrO4(2-)快，但比EuDTPA稍慢。与基于即时荧光测定的方法不同，通过使用时间分辨荧光测定法可以避免来自补充血清的培养基的自发荧光。结果表明，荧光铕螯合物为目前用于评估体外细胞细胞毒性的放射性标记物提供了一种替代方法。
