Two-state selection of conformation-specific antibodies
Two-state selection of conformation-specific antibodies
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DOI：
10.1073/pnas.0812952106
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发表时间：
2009-03-03
影响因子：
11.1
通讯作者：
Wells, James A.
中科院分区：
文献类型：
--
作者：
Gao, Junjun;Sidhu, Sachdev S.;Wells, James A.
我们提出了一种利用噬菌体展示鉴定构象特异性抗体的一般策略。使用不同的共价探针将caspase-1捕获为2种可选择的构象，称为on-form和off-form。这些构象捕获形式的蛋白酶被用作噬菌体上显示的抗体抗原结合片段（fab）的交替选择和反选择的抗原。亲和成熟后，分离出2个K-D值为2 ~ 5 nM的fab，每个fab与同源构象的结合比非同源构象紧密20 ~ 500倍。fab的动力学分析表明，结合依赖于构象，并且野生型caspase-1更接近于off-form而不是on-form。二价IgG形式的fab被用来定位细胞中的不同状态，发现活化的caspase-1集中在细胞质的中心结构中，类似于被描述为焦体的结构。这些研究证明了产生构象选择性抗体的一般策略，并显示了它们在体外和细胞中探测caspase-1构象状态分布的实用性。
We present a general strategy for identification of conformation-specific antibodies using phage display. Different covalent probes were used to trap caspase-1 into 2 alternative conformations, termed the on-form and the off-form. These conformation-trapped forms of the protease were used as antigens in alternating rounds of selection and antiselection for antibody antigen-binding fragments (Fabs) displayed on phage. After affinity maturation, 2 Fabs were isolated with K-D values ranging from 2 to 5 nM, and each bound to their cognate conformer 20- to 500-fold more tightly than their noncognate conformer. Kinetic analysis of the Fabs indicated that binding was conformation dependent, and that the wild-type caspase-1 sits much closer to the off-form than the on-form. Bivalent IgG forms of the Fabs were used to localize the different states in cells and revealed the activated caspase-1 is concentrated in a central structure in the cytosol, similar to what has been described as the pyroptosome. These studies demonstrate a general strategy for producing conformation-selective antibodies and show their utility for probing the distribution of caspase-1 conformational states in vitro and in cells.