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1.用羊抗鼠IgG(20μg/ml)包被96孔细胞培养板,再加入游离的激发型CD28单抗(终浓度为5 μg/ml);2.用激发型CD28单抗(10 μg/ml)直接包被96孔细胞培养板,然后分别加入10~5个/孔经E-花结实验获取的人外周血T细胞(PBTC),其中CD3+T>95％。逐日观察细胞的生长状态并绘制生长曲线,用3H-TdR掺入法分析PBTC的增殖效应,用FITC标记的单抗经流式细胞仪(FCM)分析细胞CD3、CD4、CD8、CTLA-4、4-1BB及OX40的表达。逐日细胞计数的结果表明,经羊抗鼠IgG包被后加入游离激发型CD28单抗或直接用激发型CD28单抗包被,均能引发PBTC的活化与增殖效应,激发3 d时的刺激指数(SI)大于9,细胞CD3、CD4、CD8、CTLA-4、4-1BB及OX40的阳性表达率(x±s,％)分别为98.6±0.2、74.4±3.1、22.5±2.0、2.1±0.4、18.4±2.2、35.2±3.5。与XGB7(作为APC)刺激的T细胞相比,经CD28单抗直接激发的T细胞,CD4+/CD8+T细胞的比值及OX40+T细胞的百分率升高(P<0.01),但不表达负性调节分子CTLA-4。
1. Coat a 96-well cell culture plate with goat anti-mouse IgG (20μg / ml) followed by free-challenged CD28 mAb (final concentration 5μg / ml); 2. Assay with stimulated CD28 monoclonal antibody / ml) were directly coated 96-well cell culture plate, and then were added to 10 ~ 5 / hole by E-flow test of human peripheral blood T cells (PBTC), CD3 + T> 95%. The cell growth status was observed daily and the growth curve was drawn. The proliferative effect of PBTC was analyzed by 3H-TdR incorporation method. The expression of CD3, CD4, CD8 and CTLA-4 was analyzed by flow cytometry (FCM) with FITC- 4-1BB and OX40 expression. The results of daily cell count showed that the activation and proliferation of PBTC could be induced by the addition of free-CD28 monoclonal antibody or goat anti-mouse CD28 antibody coated with goat anti-mouse IgG. The stimulation index (SI)> 9, the positive expression rates of CD3, CD4, CD8, CTLA-4, 4-1BB and OX40 were 98.6 ± 0.2,74.4 ± 3.1,22.5 ± 2.0,2.1 ± 0.4 , 18.4 ± 2.2, 35.2 ± 3.5. Compared with T cells stimulated with XGB7 (as APC), the ratio of CD4 + / CD8 + T cells and the percentage of OX40 + T cells directly stimulated by CD28 mAb (P <0.01) but not negative Sexual Modulator CTLA-4.