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Updated: Sep 14, 2025
Protein Engineering by Yeast Surface Display
Published on: November 29, 2024
Isolation of a Monoclonal Human scFv Against Cytomegalovirus pp71 Antigen Using Yeast Display
Kazuhisa Aoki1, Rikio Yabe1, Sayaka Ono1
1Cancer Immunology Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
Background:
Human cytomegalovirus (CMV) is a major pathogen that poses significant risks to immunocompromised individuals and neonates. The tegument protein pp71, encoded by the UL82 gene, plays a pivotal role in initiating viral lytic replication and evading host immune responses. Despite its clinical relevance, standardized monoclonal antibodies (mAbs) for pp71 remain limited, prompting the need to expand the available repertoire of antibodies targeting this critical protein.
Methods:
In this study, we constructed a diverse human single-chain variable fragment (scFv) library using RNA derived from the B cells of four healthy donors. The library was expressed in Saccharomyces cerevisiae, and iterative rounds of magnetic-activated cell sorting (MACS) were performed against recombinant pp71. Clonal enrichment was monitored using flow cytometry.
Results:
Among the isolated clones, one designated ID2 exhibited high sensitivity and specificity for pp71, as demonstrated by flow cytometry, immunofluorescence, an enzyme-linked immunosorbent assay (ELISA), and biolayer interferometry (BLI).
Conclusions:
Collectively, these findings establish a novel pp71-specific mAb and underscore the utility of yeast surface display combined with MACS for expanding the antibody toolkit available for CMV research and diagnostics.
Insights
Researchers developed a new monoclonal antibody (mAb) targeting human cytomegalovirus (CMV) tegument protein pp71. This advancement expands the toolkit for CMV research and diagnostics.
Area of Science:
- Immunology
- Virology
- Biotechnology
Background:
- Human cytomegalovirus (CMV) is a significant pathogen, particularly for immunocompromised individuals and neonates.
- The CMV tegument protein pp71 (UL82) is crucial for viral replication and immune evasion.
- Limited availability of standardized monoclonal antibodies (mAbs) against pp71 hinders CMV research and diagnostics.
Purpose of the Study:
- To develop novel monoclonal antibodies (mAbs) targeting the human cytomegalovirus (CMV) tegument protein pp71.
- To address the need for expanded antibody tools for CMV research and clinical applications.
Main Methods:
- Construction of a diverse human single-chain variable fragment (scFv) library from healthy donor B cells.
- Expression of the scFv library in Saccharomyces cerevisiae for yeast surface display.
- Iterative magnetic-activated cell sorting (MACS) against recombinant pp71, with clonal enrichment monitored by flow cytometry.
Main Results:
- Isolation of a specific clone, designated ID2, with high sensitivity and specificity for pp71.
- Validation of ID2's performance using flow cytometry, immunofluorescence, ELISA, and biolayer interferometry (BLI).
Conclusions:
- Establishment of a novel pp71-specific mAb.
- Demonstration of yeast surface display combined with MACS as an effective strategy for antibody discovery.
- Contribution to the expansion of the antibody repertoire for CMV research and diagnostics.
