Two-state selection of conformation-specific antibodies

Area of Science:

• Immunology
• Biochemistry
• Molecular Biology

Background:

• Antibodies typically recognize specific epitopes on antigens.
• Conformational changes in proteins can alter their function and antigenicity.
• Developing tools to differentiate protein conformational states is crucial for understanding biological processes.

Purpose of the Study:

• To establish a general strategy for generating conformation-specific antibodies.
• To isolate antibodies that selectively bind to distinct conformations of caspase-1.
• To utilize these antibodies to investigate caspase-1 localization in cellular contexts.

Main Methods:

• Phage display technology was employed for antibody selection.
• Covalent probes were used to trap caspase-1 into 'on' and 'off' conformations.
• Alternating rounds of selection and anti-selection were performed against these trapped conformations.
• Affinity maturation was used to enhance antibody binding affinity.

Main Results:

• Two high-affinity (2-5 nM K(D)) conformation-specific antibody fragments (Fabs) were isolated.
• These Fabs exhibited 20- to 500-fold greater binding to their cognate conformer compared to the non-cognate one.
• Kinetic analysis confirmed conformation-dependent binding, with wild-type caspase-1 favoring the 'off-form'.
• Activated caspase-1 was localized to a central cytosolic structure, resembling a pyroptosome.

Conclusions:

• A versatile phage display strategy for producing conformation-selective antibodies was demonstrated.
• The isolated antibodies are valuable tools for distinguishing caspase-1 conformational states.
• The findings provide insights into the cellular localization and distribution of activated caspase-1.