Selection and optimization strategy for Rap1-targeting single-domain antibodies as platelet activation markers.

ABSTRACT:

Background

Rap1 is critical for platelet activation, functioning as a key node of the platelet activation pathways.

Objectives

This study aimed to develop VHH-Fc (minibodies) against Rap1 for the purpose to quantify active Rap1 levels in platelets.

Methods

We have produced the first generation of VHHs against active Rap1 through a series of negative and positive screenings of a synthetic phage display library, utilizing both inactive and active Rap1B. We performed random mutagenesis, followed by yeast 2-hybrid screening to optimize variants.

Results

Among 122 VHH clones, 2 with the highest redundancy were subcloned as VHH-Fc. Both selectively detected active Rap1B G12V in HeLa cells but failed to recognize the inactive Rap1B S17N isoform. They successfully capt

SUBMITTER: Alessi MC 

PROVIDER: S-EPMC12860359 | biostudies-literature | 2026 Jan

REPOSITORIES: biostudies-literature