Bouma G J, van der Meer-Prins P M, van Bree F P, van Rood J J, Claas F H
Department of Immunohematology, Leiden University Hospital, The Netherlands.
Hum Immunol. 1992 Oct;35(2):85-92. doi: 10.1016/0198-8859(92)90015-f.
We report on the use of europium (Eu) as a suitable nonradioactive alternative for target cell labeling in limiting dilution analysis (LDA) assays set up to determine cytotoxic T-lymphocyte precursor (CTLp) frequencies. A nonradioactive alternative to the commonly used chromium-51 (51Cr) release assay seems desirable because working with radioisotopes has some major disadvantages concerning possible health risks, environmental load, costs of facilities necessary for working with radioisotopes, and shelf life. Some groups have successfully applied the Eu release assay based on detection by time-resolved fluorometry, to tests in which NK- or LAK-cell activity or cytotoxic T-lymphocyte reactions were measured. This led to the investigation whether this method could also be applicable to the more specific determination of CTLp frequencies in LDA assays. After optimal labeling conditions had been established, the sensitivity of the Eu release assay was determined by performing several LDA assays in which the target cells were labeled with either Eu or radioactive 51Cr. When CTLp frequencies were compared, it was shown that the Eu release assay is at least as sensitive and specific as the 51Cr release assay. Moreover, although the labeling procedure takes longer, sample processing is much faster: only 1 second per sample. The fact that the Eu release assay is not radioactive enables the assay to be performed at any laboratory and even--because the frequency of CTLps may have implications for organ graft survival and for donor selection in bone marrow transplantation--to do so on a routine basis.
我们报告了在为确定细胞毒性T淋巴细胞前体（CTLp）频率而设立的有限稀释分析（LDA）试验中，使用铕（Eu）作为靶细胞标记的合适非放射性替代物。对于常用的铬-51（51Cr）释放试验而言，一种非放射性替代物似乎是可取的，因为使用放射性同位素存在一些重大缺点，涉及可能的健康风险、环境负荷、使用放射性同位素所需设施的成本以及保质期。一些研究小组已成功将基于时间分辨荧光测定法检测的Eu释放试验应用于测量NK或LAK细胞活性或细胞毒性T淋巴细胞反应的试验中。这引发了对于该方法是否也可应用于更具体地测定LDA试验中CTLp频率的研究。在确定了最佳标记条件后，通过进行若干LDA试验来测定Eu释放试验的灵敏度，其中靶细胞分别用Eu或放射性51Cr进行标记。当比较CTLp频率时，结果表明Eu释放试验至少与51Cr释放试验一样灵敏和特异。此外，尽管标记过程耗时更长，但样品处理速度要快得多：每个样品仅需1秒。Eu释放试验无放射性这一事实使得该试验能够在任何实验室进行，甚至——由于CTLp的频率可能对器官移植存活以及骨髓移植中的供体选择有影响——能够作为常规操作进行。
