Site Specific Fluorescent Labeling via SpyTag SpyCatcher for Rapid Hybridoma Screening in Semi-Solid Medium

Conventional hybridoma screening in semi-solid medium relies on antigens tagged with visible fluorophores, such as FITC or AF488, to quickly recognize antibody-producing cell lines. Regrettably, traditional chemical bonding methods involving NHS-esters or isothiocyanate groups often alter lysine residues within the targeted epitopes, potentially disrupting antibody recognition of these vital areas.

In this report, researchers detail a SpyTag SpyCatcher-based site-specific labeling approach that avoids antigen modification during screening. The innovative technique involves genetically attaching a 16-amino-acid SpyTag to the C-terminus of the target antigen, facilitating covalent linking to an sfGFP SpyCatcher fluorescent probe.

When placed in semi-solid medium alongside both the SpyTag-antigen and sfGFPSpyCatcher, positive hybridoma clones become easily identifiable through unique fluorescent halos. Conversely, negative clones do not exhibit any detectable signal.

Remarkably, the site-specific methodology resulted in a substantially higher proportion of fluorescence-positive clones compared to the conventional AF488-labeled antigen technique. This implies that preserving the epitope structure contributes to improved screening recovery rates. Importantly, the site-specific approach did not negatively impact hybridoma growth or final clone positivity, presenting a straightforward, speedy, and epitope-friendly method for monoclonal antibody screening within semi-solid medium.