The V5-Epitope Tag for Cell Engineering and Its Use in Immunohistochemistry and Quantitative Flow Cytometry

Area of Science:

• Synthetic Biology
• Molecular Biology
• Immunology

Background:

• Synthetic biology enables advanced cell engineering and therapeutics like CAR-T cells.
• Accurate detection, localization, and quantification of fusion proteins are crucial for these applications.
• The V5 tag is a promising epitope tag for such purposes but is underutilized.

Purpose of the Study:

• To systematically evaluate the murine (mu_SV5-Pk1) and humanized (hu_SV5-Pk1) anti-V5 tag antibodies.
• To assess the V5 tag's suitability for detecting V5-tagged receptors in various experimental settings.
• To optimize immunohistochemistry (IHC) for V5-tagged cells in fixed tissues.

Main Methods:

• Evaluation of anti-V5 antibodies (mu_SV5-Pk1 and hu_SV5-Pk1) in vitro and in vivo.
• Assessment of fixation and detachment reagent effects on V5 tag signal.
• Immunohistochemistry (IHC) on formalin-fixed paraffin-embedded (FFPE) mouse and human tissues.

Main Results:

• The V5 tag signal is sensitive to specific fixation and detachment reagents.
• The humanized antibody (hu_SV5-Pk1) improved IHC by reducing cross-reactivity and background noise in mouse tissues.
• The murine antibody (mu_SV5-Pk1) demonstrated no unspecific binding in 46 human tissue samples.

Conclusions:

• The V5 tag and its cognate antibodies provide a robust tool for synthetic biology.
• Optimized antibody selection and IHC protocols enhance V5-tagged protein detection in diverse biological samples.
• This work facilitates the application of V5-tagged proteins in cell engineering and therapeutic development.