Application of protein A-rosette assay for screening of monoclonal antibodies to human complement regulatory proteins.
Application of protein A-rosette assay for screening of monoclonal antibodies to human complement regulatory proteins.
复制标题
应用蛋白 A 花环测定法筛选人补体调节蛋白的单克隆抗体。
DOI：
10.1159/000463132
复制
发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Hitoshi Akedo
中科院分区：
文献类型：
--
作者：
Tsukasa Seya;Tomoko Hara;A. Uenaka;Eiichi Nakayama;Hitoshi Akedo
用纯化的膜辅因子蛋白（MCP）免疫小鼠，用蛋白A（PA）花环试验筛选其单克隆抗体。在该试验中，将杂交瘤细胞的培养上清液覆盖在固定的携带MCP的细胞上，洗涤后，将PA包被的绵羊红细胞作为指示剂应用于这些携带MCP的细胞。因此，在整个筛选过程中不需要纯化抗原。在6 h内成功检测了300多个收获的上清液。每个所得抗体由IgG的单一亚类组成，并且仅在transblotted和表面标记的材料中与MCP反应。然后用这些纯化的抗体评估该测定的灵敏度。发现可检测到低至0.5 μ g的IgG 1或0.01 μ g的IgG 2a，且玫瑰花结形成超过30%。有变化的细胞系之间的敏感性PA-玫瑰花结试验和敏感性不相关的MCP表面表达的数量在任何细胞系。K562给出了最低的背景（非特异性玫瑰花结形成）和最好的特异性抗MCP的20个MCP阳性细胞系测试。对适合检测单克隆抗衰变加速因子和抗C3 b/C4 b受体的细胞系进行了检查，发现CCRF-SB和HSB 2以及外周血粒细胞分别是筛选衰变加速因子和C3 b/C4 b受体的合适细胞系。通过PA-玫瑰花结试验，使用粒细胞成功地获得了抗C3 b/C4 b受体的克隆。该方法不需要纯化抗原，有利于快速筛选和纯化抗细胞表面补体调节蛋白的阳性克隆。
Mice were immunized with purified membrane cofactor protein (MCP) and its monoclonal antibodies were screened by protein A(PA)-rosette assay. In this assay, the culture supernatants of hybridoma cells were layered over fixed MCP-bearing cells, and after washing, PA-coated sheep erythrocytes were applied as an indicator to these MCP-bearing cells. No purified antigen was therefore required throughout the screening. More than 300 of the supernatants harvested were successfully examined within 6 h. Each resultant antibody consisted of a single subclass of IgG, and reacted only with MCP in both transblotted and surface-labeled materials. The sensitivity of this assay was then assessed with these purified antibodies. As little as 0.5 micrograms of IgG1 or 0.01 micrograms of IgG2a was found to be detectable with more than 30% rosette formation. There were variations among cell lines in the sensitivity to the PA-rosette assay and the sensitivity did not correlate with the quantity of MCP surface expression in any of the cell lines. K562 gave the lowest background (nonspecific rosette formation) and the best specificity for anti-MCP of the 20 MCP-positive cell lines tested. Cell lines suitable for the detection of monoclonal anti-decay-accelerating factor and anti-C3b/C4b receptor were also examined and CCRF-SB and HSB2, and peripheral blood granulocytes, were found to be proper cell lines for screening the decay-accelerating factor and C3b/C4b receptor, respectively. Clones for anti C3b/C4b receptor were successfully obtained using granulocytes by the PA-rosette assay. This method needs no purified antigen and facilitates the rapid screening and purification of positive clones against cell-surface complement regulatory proteins.