Isolation of cell surface-specific human monoclonal antibodies using phage display and magnetically-activated cell sorting: applications in immunohematology
Isolation of cell surface-specific human monoclonal antibodies using phage display and magnetically-activated cell sorting: applications in immunohematology
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DOI：
10.1016/s0022-1759(97)00087-2
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发表时间：
1997-08-07
影响因子：
2.2
通讯作者：
Bunya, VY
中科院分区：
文献类型：
--
作者：
Siegel, DL;Chang, TY;Bunya, VY
描述了一种分离丝状噬菌体展示的针对不可纯化的细胞表面表达分子的人单克隆抗体的方法。为了优化抗原特异性噬菌体的捕获并使不相关噬菌体抗体的结合最小化，采用同时的阳性和阴性选择策略。将携带目标抗原的细胞用磁珠预包被并稀释到过量的未修饰的抗原阴性细胞中。将细胞混合物与Fab/噬菌体文库孵育后，使用磁激活细胞分选回收抗原阳性细胞群，并洗脱抗原特异性Fab/噬菌体并在细菌培养物中增殖。利用该方案，使用磁性标记的Rh（D）阳性和过量未标记的Rh（D）阴性人红细胞和从人外周血淋巴细胞构建的Fab/噬菌体文库，从单个同种异体免疫个体中分离出数十种独特的临床有用的γ（1）κ和γ（1）λ抗Rh（D）抗体。这种细胞表面选择方法很容易适用于其它系统，例如用于鉴定推定的肿瘤特异性抗原，并提供了快速（
A method is described for the isolation of filamentous phage-displayed human monoclonal antibodies directed at unpurifiable cell surface-expressed molecules. To optimize the capture of antigen-specific phage and minimize the binding of irrelevant phage antibodies, a simultaneous positive and negative selection strategy is employed. Cells bearing the antigen of interest are pre-coated with magnetic beads and diluted into an excess of unmodified antigen-negative cells. Following incubation of the cell admixture with a Fab/phage library, the antigen-positive cell population is retrieved using magnetically-activated cell sorting and antigen-specific Fab/phage are eluted and propagated in bacterial culture. Utilizing this protocol with magnetically-labeled Rh(D)-positive and excess unlabeled Rh(D)-negative human red blood cells and a Fab/phage library constructed from human peripheral blood lymphocytes, dozens of unique clinically-useful gamma(1) kappa and gamma(1) lambda anti-Rh(D) antibodies were isolated from a single alloimmunized individual. This cell-surface selection method is readily adaptable for use in other systems, such as for the identification of putative tumor-specific antigens and provides a rapid (