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本文建立的免疫亲合柱纯化细胞的方法,其基本原理是利用亲合素交联的凝胶捕获生物素化单抗阳性细胞,去除非目的细胞、利用该柱进行了CD8~+细胞的纯化实验,证明此方法获得的细胞纯度高,回收率好,且操作简便易行.将抗CD8单克隆抗体经DEAE离子交换色谱纯化,在碳二亚胺盐酸盐作用下,与生物素化N-羟基琥珀酰亚氨酯交联,制备生物素化抗CD8单克隆抗体.将Avidin与Bio-Gel利用化学法交联并装于4ml软塑料柱内,用PBS充分冲洗后,以5%BSA平衡柱子.用常规方法制备外周血单个核细胞(PBMNC).将PBMNC调整细胞浓度为5×10~7～2×10~8/ml,加入约1/10体积的生物素化抗CD8单克隆抗体,4℃温育30min.用含1%BSA的PBS洗细胞1次,并将细胞悬于2ml5%BSA中,加细胞于柱子上,使细胞缓缓流入凝胶内,静置约3～5min.先以5%BSA洗去未吸附的细胞,再以PBS洗去BSA,用手轻轻挤压柱子,CD8~+细胞即与凝胶脱离,随PBS流出柱子,收集CD8~+细胞进行检测.
The method of immunoaffinity column purification in this paper is based on the principle of capturing the biotinylated monoclonal antibody positive cells with avidin crosslinked gel, removing the non-target cells and purifying the CD8 + cells by using the column Experiments show that the method obtained by the cell purity, good recovery, and easy to operate.The anti-CD8 monoclonal antibody by DEAE ion exchange chromatography purification, in the carbodiimide hydrochloride, biotinylated N -hydroxysuccinimide cross-link to prepare biotinylated anti-CD8 mAb Avidin and Bio-Gel were chemically cross-linked and mounted in 4 ml soft plastic column, washed well with PBS, washed with 5% BSA The column was equilibrated and peripheral blood mononuclear cells (PBMNC) were prepared by a conventional method. PBMNCs were adjusted to a cell concentration of 5 × 10 -7 to 2 × 10 -8 / ml, and about 1/10 volume of biotinylated anti-CD8 monoclonal The antibody was incubated for 30 min at 4 ° C. The cells were washed once with PBS containing 1% BSA and suspended in 2 ml of 5% BSA. Cells were added to the column and allowed to flow slowly into the gel. 5min. The non-adsorbed cells were washed with 5% BSA, then BSA was washed with PBS, and the column was gently squeezed by hand. The CD8 ~ + cells were detached from the gel and eluted with PBS Column, collecting CD8 ~ + cells were detected.