一种简便的固相单抗富集CD4^+细胞方法

A simple method for enriching CD4^+ cells from peripheral blood mononuclear cells using solid phase anti-CDs monoclonal antibodies

目的 探索建立一种富集CD4 + 细胞的方法 ,藉以提高免疫细胞化学法计数Th1/Th2细胞的准确性。方法 取 2 1份健康人外周抗凝血 ,无菌条件下分离单个核细胞 (PBMC) ,用CD4McAb包被 2 4孔细胞培养板 ,加入PBMC ,温育后吸弃未结合细胞 ,继而吸打使结合于板上的细胞脱落 ,收集细胞 (阳性选择 ) ;用CD8McAb、CD16McAb、WuBMcAb包被 2 4孔细胞培养板 ,加入PBMC ,温育后吸取未结合细胞 (阴性选择 )。用生物素 链霉亲和素免疫细胞化学法检测富集前及用两种选择方法富集所得细胞中CD4 + 细胞 ,计算百分率、活细胞率和CD4 + 细胞回收率。结果 富集前 2 1份样品的PBMC中CD4 + 细胞百分率 4 3.7%± 3.1% ,经阳性选择和阴性选择后CD4 + 细胞百分率分别为 90 .9%± 2 .8%和 76 .2 %± 2 .5 % ,经卡方检验 ,x2 >x20 .0 0 1,差异有非常显著性 ,阳性选择富集的CD4 + 细胞纯度显著高于阴性选择。经阳性选择和阴性选择后 ,CD4 + 细胞回收率分别为 6 4 .3%± 4 .9%和 6 9.7%± 5 .2 % ,经卡方检验 ,x2 >x20 .0 0 1,差异有非常显著性 ,阴性选择法的细胞回收率明显高于阳性选择法。富集后活细胞率保持在 95 %以上。结论 所建立的固相单抗方法可有效富集CD4 + 细胞并保持很高的活细胞率 ,其中阳性选择是首选方法 ;CD4 + 细?

Objective To establish a method for enriching CD4 cells from peripheral blood mononuclear cells in order to account Th1/Th2 cells more accurately by immunocytochemical method. Method Heparinized blood samples were obtained from 21 healthy donors. Peripheral blood mononuclear cells(PBMCs) were isolated by standard procedure. The PBMCs were put in the wells of 24 wells flat bottom cell culture plate coated with anti CD4 monoclonal antibody (McAb) (or anti CD8, anti CD16 and anti B McAb), the cells were collected using positive or negative selection method respectively. The CD4 + cells before and after enrichment were detected by biotin streptavidin immunocytochemical method. The cells were stained by trypan blue. The percentage of CD4 + cells, percentage of living cells and rate of recovery of CD4 + cells were calculated. Result The percentage of CD4 + cells of PBMCs from 21 donors was 43.7±3.1% before enrichment and the percentages of CD4 + cells after enrichment by positive and negative selection method were 90.9±2.8% and 76.2±2.5% respectively, there was significant difference; the rate of recovery by positive and negative selection method were 64.3±4.9% and 69.7±5.2% respectively, there was also significant difference; the percentage of living cells was over 95%. Conclusion The method for enriching CD4 cells from peripheral blood mononuclear cells using solid phase anti CDs McAb is effective and the enriched cells have high vitality. The positive selection method among enriching methods is favoured. The enrichment of CD4 is the essential pretreating procedure for counting Th1/Th2 cells by immunocytochemical method.