SELECTION OF PHAGE ANTIBODIES BY BINDING-AFFINITY - MIMICKING AFFINITY MATURATION
SELECTION OF PHAGE ANTIBODIES BY BINDING-AFFINITY - MIMICKING AFFINITY MATURATION
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DOI：
10.1016/0022-2836(92)90639-2
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发表时间：
1992-08-05
影响因子：
5.6
通讯作者：
WINTER, G
中科院分区：
文献类型：
--
作者：
HAWKINS, RE;RUSSELL, SJ;WINTER, G
我们描述了一种基于丝状噬菌体表面抗体展示的方法，用于通过其对抗原的亲和力或通过其从抗原解离的动力学（解离速率）来选择抗体。对于亲和力选择，将噬菌体与少量可溶性生物素化抗原（< 1 μg）混合，使得抗原超过噬菌体，但抗原浓度低于抗体的解离常数（Kdd）。然后使用链霉亲和素包被的顺磁珠选择那些与抗原结合的噬菌体。该过程可以区分具有密切相关亲和力的抗体。对于解离速率选择，将抗体预加载有生物素化的抗原，并在链霉亲和素包被的顺磁珠上捕获之前稀释至过量的未标记抗原中可变时间。为了模拟免疫系统的亲和力成熟过程，我们在体外使用易错聚合酶将随机突变引入抗体基因，并使用亲和力选择来分离具有改进亲和力的突变体。从小鼠抗体B1.8的突变体（每个VH基因平均1.7个碱基变化）的小文库（40，000个克隆）开始，并使用几轮亲和力选择，我们分离出对半抗原4-羟基-5-碘-3-硝基-苯乙酰基-（NIP）-己酸具有四倍提高的亲和力的突变体（与B1.8Kd = 41.9（±1.6）nM相比，μ M Kd = 9.4（±0.3）nM）。突变体亲和力的相对增加与抗-4-羟基-3-硝基苯乙酰基/NIP-己酸小鼠二次免疫应答中观察到的增加相当。
We describe a process, based on display of antibodies on the surface of filamentous bacteriophage, for selecting antibodies either by their affinity for antigen or by their kinetics of dissociation (off-rate) from antigen. For affinity selection, phage are mixed with small amounts of soluble biotinylated antigen (< 1 μg) such that the antigen is in excess over phage but with the concentration of antigen lower than the dissociation constant (Kdd) of the antibody. Those phage bound to antigen are then selected using streptavidin-coated paramagnetic beads. The process can distinguish between antibodies with closely related affinities. For off-rate selection, antibodies are preloaded with biotinylated antigen and diluted into excess unlabelled antigen for variable times prior to capture on streptavidincoated paramagnetic beads. To mimic the affinity maturation process of the immune system, we introduced random mutations into the antibody genesin vitrousing an error-prone polymerase, and used affinity selection to isolate mutants with improved affinity. Starting with a small library (40,000 clones) of mutants (average 1.7 base changes per VHgene) of the mouse antibody B1.8, and using several rounds of affinity selection, we isolated a mutant with a fourfold improved affinity to the hapten 4-hydroxv-5-iodo-3-nitro-phenacetyl-(NIP)-caproic acid (mutantKd= 9.4(±0.3)nMcompared withBl.8Kd= 41.9(±1.6)nM). The relative increase in affinity of the mutant is comparable to the increase seen in the anti-4-hydroxy-3 nitrophenylacetyl/NIP-caproic acid murine secondary immune response.