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Direct Antibody Isolation on Cells Using Affinity-Tag-Guided Proximity Selection.

You-Chiun Chang1, Chia-Yi Kao1,2, Hao-Cheng Tang1

  • 1Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.

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Researchers developed affinity-tag-guided proximity selection (A-GPS), a novel method for generating specific antibodies against difficult membrane proteins without antigen purification. This technique significantly improves antibody isolation efficiency and specificity for therapeutic applications.

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Area of Science:

  • Biotechnology
  • Immunology
  • Molecular Biology

Background:

  • Traditional antibody generation methods require purified antigens, posing challenges for membrane proteins like GPCRs, SLCs, and ion channels, which are crucial drug targets.
  • Whole-cell panning offers an alternative but is hampered by background noise and low success rates, necessitating extensive screening.

Purpose of the Study:

  • To develop an efficient method for isolating specific antibodies against challenging membrane proteins directly from antigen-presenting cells.
  • To overcome the limitations of traditional antibody generation and whole-cell panning by reducing background interference and increasing success rates.

Main Methods:

  • Introduced affinity-tag-guided proximity selection (A-GPS), a phage selection strategy that genetically fuses an affinity tag to the target antigen.
  • A-GPS confines proximity labeling near the target antigen on cells, enriching phages with high affinity and specificity.
  • Validated A-GPS using surface-presented GFP on human cells, performing two rounds of selection.

Main Results:

  • A-GPS successfully enriched antigen-specific phage clones in just two rounds.
  • Over 95% of randomly selected clones exhibited high affinity and specificity for GFP, outperforming background HEK293T surface proteins.
  • A derived Fab fragment demonstrated subnanomolar binding affinity for GFP and proved effective in immunofluorescence and flow cytometry.

Conclusions:

  • A-GPS is a highly efficient and specific method for generating antibodies against difficult-to-purify antigens, particularly membrane proteins.
  • The developed A-GPS technique facilitates the production of commercial-grade antibodies suitable for various biological applications.
  • This method offers a significant advancement for antibody discovery and development in drug discovery and biotechnology.