Related Experiment Video
Updated: Apr 21, 2026
Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood
Published on: February 6, 2018
Generation of a single chain antibody variable fragment (scFv) to sense selectively RhoB activation
Patrick Chinestra1, Aurélien Olichon2, Claire Medale-Giamarchi2
1Inserm, UMR 1037-CRCT, GTPases Rho dans la progression tumorale, Toulouse, France.
Abstract:
Determining the cellular level of activated form of RhoGTPases is of key importance to understand their regulatory functions in cell physiopathology. We previously reported scFvC1, that selectively bind to the GTP-bound form of RhoA, RhoB and RhoC. In this present study we generate, by molecular evolution, a new phage library to isolate scFvs displaying high affinity and selectivity to RhoA and RhoB. Using phage display affinity maturation against the GTP-locked mutant RhoAL63, we isolated scFvs against RhoA active conformation that display Kd values at the nanomolar range, which corresponded to an increase of affinity of three orders of magnitude compared to scFvC1. Although a majority of these evolved scFvs remained selective towards the active conformation of RhoA, RhoB and RhoC, we identified some scFvs that bind to RhoA and RhoC but not to RhoB activated form. Alternatively, we performed a substractive panning towards RhoB, and isolated the scFvE3 exhibiting a 10 times higher affinity for RhoB than RhoA activated forms. We showed the peculiar ability of scFvE3 to detect RhoB but not RhoA GTP-bound form in cell extracts overexpressing Guanine nucleotide Exchange Factor XPLN as well as in EGF stimulated HeLa cells. Our results demonstrated the ability of scFvs to distinguish RhoB from RhoA GTP-bound form and provide new selective tools to analyze the cell biology of RhoB GTPase regulation.
Insights
Researchers developed new high-affinity antibodies (scFvs) to detect active Rho GTPases. These tools can specifically identify the GTP-bound form of RhoB, aiding cell biology research.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Determining the active, GTP-bound state of Rho GTPases is crucial for understanding cellular processes and diseases.
- Previous tools, like scFvC1, showed limited selectivity for specific Rho GTPase isoforms.
- There is a need for high-affinity and isoform-specific antibodies to study Rho GTPase regulation.
Purpose of the Study:
- To generate and isolate novel single-chain variable fragments (scFvs) with enhanced affinity and selectivity for active RhoA and RhoB.
- To develop tools capable of distinguishing between different Rho GTPase isoforms in their active states.
- To provide new reagents for analyzing Rho GTPase cell biology.
Main Methods:
- Phage display technology and molecular evolution were employed to create new antibody libraries.
- Affinity maturation techniques, including panning against GTP-locked RhoA mutants (RhoAL63), were used to isolate high-affinity scFvs.
- Subtractive panning against RhoB was performed to achieve isoform-specific antibody isolation.
Main Results:
- Evolved scFvs demonstrated nanomolar affinity for the active conformation of RhoA, a significant improvement over previous antibodies.
- Most isolated scFvs remained selective for the active forms of RhoA, RhoB, and RhoC.
- A specific scFv (scFvE3) was identified with 10-fold higher affinity for active RhoB than active RhoA.
- scFvE3 successfully detected GTP-bound RhoB but not GTP-bound RhoA in cellular extracts under various conditions.
Conclusions:
- Novel scFvs have been developed with significantly improved affinity and selectivity for active Rho GTPases.
- scFvE3 provides a unique tool for specifically detecting the active form of RhoB, distinguishing it from RhoA.
- These new reagents offer valuable capabilities for detailed analysis of RhoB GTPase regulation in cell biology.
