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Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3071-6. doi: 10.1073/pnas.0812952106. Epub 2009 Feb 10.

Two-state selection of conformation-specific antibodies.

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  • 1Department of Pharmaceutical Chemistry, University of California, 1700 4th Street, San Francisco, CA 94143, USA.

Abstract

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.

PMID:
19208804
[PubMed - indexed for MEDLINE]
PMCID:
PMC2651286
Free PMC Article
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