摘要
目的: 建立一种新的单细胞内IFN -γ的检测方法毛细管电泳免疫分析激光诱导荧光法 (CEIA- LIF)。方法:选 2例重型再障(severaplasticanemia, SAA)患者及 1例正常人, 用免疫磁珠法分离纯化外周血CD8+ T细胞。将其与毛地黄皂苷反应 15min, 再与FITC 抗IFN -γ单抗 (mAb)反应20min。用CEIA -LIF法进行单个细胞的连续检测, 并用倒置显微镜及激光扫描共聚焦荧光显微镜等证实本法的可行性。结果: 纯化的CD8+ T细胞内的IFN γ, 是在不溶膜的情况下得到全部检测。2例SAA患者外周血单个CD8+ T细胞内IFN -γ的含量, 分别为 ( 151. 53±28. 92 )zmol和 ( 223. 72±45. 23)zmol, 高出正常对照 ( 47. 47±17. 97 )zmol约 3 ~4倍(P<0. 01)。结论: CEIA LIF法对单个CD8+ T细胞内IFN -γ含量的测定是可行的, 有望用于临床检测。
AIM: To establish a new method capillary electrophoretic immunoassay laser-induced fluorescence (CEIA-LIF), to detect IFN-γ level in single CD8+ T cell. METHODS: CD8+ T cells were isolated from peripheral blood of two patients with severe aplastic anemia(SAA) and one normal person by Ficoll-Hypaque gradient centrifugation and then were purified by immunomagnetic microbead separation. Then purified CD8+ T cells were incubated with digitonin for 15 min followed by FITC-anti-IFN-γ mAb for 20 min. The single cell was detected continuously by CEIA-LIF. The feasibility of the method was confirmed by inverted microscopy and laser scanning confocal fluorescence microscopy. RESULTS: The IFN-γ content in purified CD8+ T cells was detected under the condition of cell-membrane integrity. The IFN-γ level in single CD8+ T cell from 2 SAA patients was (151.53±28.92)zmol and (223.72±45.23)zmol, respectively, and much higher that from normal control (47.47±17.97)zmol (P=0.001). CONCLUSION: It is feasible to quantitate IFN-γ in single CD8+ T cell by CEIA-LIF. CEIA-LIF might be useful in the clinical detection of intracellular cytokines.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2005年第1期72-75,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
山东省自然科学基金资助项目(No.Y2001C09)
