Site-Specific Fluorescent Labeling via SpyTag-SpyCatcher for Rapid Hybridoma Screening in Semi-Solid Medium
Year: 2026
Type: preprint
Abstract: Hybridoma screening in semi-solid medium typically employs antigens labeled with visible fluorophores (e.g., FITC, AF488) to enable single-step identification of antibody-secreting clones. However, conventional chemical conjugation via NHS-esters or isothiocyanate groups frequently modifies lysine residues located within epitopes, potentially abrogating antibody recognition of these critical regions. Here, we describe a SpyTag–SpyCatcher-based site-specific labeling strategy that circumvents epitope damage during semi-solid medium screening. A 16-amino-acid SpyTag was genetically fused to the C-terminus of the target antigen, enabling covalent conjugation to an sfGFP–SpyCatcher fluorescent probe. In semi-solid medium supplemented with SpyTag-antigen and sfGFP–SpyCatcher, positive hybridoma clones were readily identified by distinct fluorescent halos, whereas negative clones showed no detectable signal. Notably, the site-specific method yielded a significantly higher frequency of fluorescence-positive clones compared to the conventional AF488-labeled antigen method, suggesting that epitope preservation enhances screening recovery. Furthermore, this approach did not impair hybridoma growth or final clone positivity, offering a simple, rapid, and epitope-compatible method for monoclonal antibody screening.
Authors: Amber Guo, Moris Wei, Jonny Wu, Xiaoxiao Li, Bruce Jiang
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7204806270