Abstract：
:Traditional antibody generation, using either phage display or animal immunization, relies on purified antigens. Many membrane proteins, such as G protein-coupled receptors, solute carriers, or ion channels, are important drug targets but very challenging for the formation of antibodies due to the difficulty of protein purification. Whole-cell panning is an alternative approach for generating antibodies without the need for antigen purification. However, it often suffers from background interference and therefore requires extensive screening with low success rates. Here, we develop a new phage selection method, dubbed affinity-tag-guided proximity selection (A-GPS), to efficiently isolate specific antibodies directly from the antigen-presenting cells. By engineering a genetically fused affinity tag for the target antigen, A-GPS confines the proximity labeling reaction near the target antigen and preferentially enriches the phage bound to the target antigen. Using surface-presented GFP on human cells as a model antigen, we demonstrated that A-GPS successfully enriched the antigen-specific clones in two rounds of selection. Among the 46 randomly picked clones, >95% of clones showed great affinity and specificity for GFP over the background of HEK293T surface proteins. One of the best clones expressed as a Fab fragment showed subnanomolar binding affinity for GFP. This clone was successfully applied to common biological applications, such as immunofluorescence and flow cytometry, reflecting the usefulness of A-GPS for generating commercial-grade antibodies.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Chang YC,Kao CY,Tang HC,Huang MS,Mou KYdoi
10.1021/acs.biochem.0c00730subject
Has Abstractpub_date
2020-11-10 00:00:00pages
4285-4293issue
44eissn
0006-2960issn
1520-4995journal_volume
59pub_type
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