Two-state selection of conformation-specific antibodies Export

@article{citeulike:4281160, abstract = {10.1073/pnas.0812952106 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 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.}, author = {Gao, Junjun and Sidhu, Sachdev S. and Wells, James A.}, citeulike-article-id = {4281160}, citeulike-linkout-0 = {http://dx.doi.org/10.1073/pnas.0812952106}, citeulike-linkout-1 = {http://www.pnas.org/content/106/9/3071.full.abstract}, citeulike-linkout-2 = {http://www.pnas.org/content/106/9/3071.full.full.pdf}, citeulike-linkout-3 = {http://view.ncbi.nlm.nih.gov/pubmed/19208804}, citeulike-linkout-4 = {http://www.hubmed.org/display.cgi?uids=19208804}, day = {3}, doi = {10.1073/pnas.0812952106}, journal = {Proceedings of the National Academy of Sciences}, keywords = {allostery, dimerization, experiment, protein, structural\_change}, month = {March}, number = {9}, pages = {3071--3076}, posted-at = {2009-04-07 08:27:35}, priority = {2}, title = {Two-state selection of conformation-specific antibodies}, url = {http://dx.doi.org/10.1073/pnas.0812952106}, volume = {106}, year = {2009} }

TY - JOUR ID - citeulike:4281160 L3 - citeulike-article-id:4281160 N2 - 10.1073/pnas.0812952106 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 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. IS - 9 JF - Proceedings of the National Academy of Sciences EP - 3076 TI - Two-state selection of conformation-specific antibodies VL - 106 SP - 3071 KW - allostery KW - dimerization KW - experiment KW - protein KW - structural_change AU - Gao, Junjun AU - Sidhu, Sachdev AU - Wells, James PY - 2009/03/03/ UR - http://dx.doi.org/10.1073/pnas.0812952106 ER -