Use of a yeast display library of human antibody recognition sequences to develop detection assays for potential cancer biomarkers.
Barbara Garvik, Nicole Urban, Nathalie Scholler
Public Health Sciences, Fred Hutchinson Cancer Rsh Ctr, 1100 Fairview Ave. N, Seattle, WA, 98109, USA
Libraries of antibody recognition sequences displayed on the surface of bacteriophage or yeast represent an alternative to the production of monoclonal antibodies by animal immunization and hybridoma technology. Isolation of antibody fragments with the desired specificity does not rely upon the immunogenicity of the target. Other advantages are speed and low cost. We are using the yeast surface display library developed by the Pacific Northwest National Laboratory (Feldhaus et al., Nat Biotechnol 2003, 163-70) to create an ELISA test to measure HE4, a protein elevated in the serum of many ovarian cancer patients. We produced a sublibrary of yeast expressing single-chain fragment variable regions (scFv) specific for HE4 by sequential enrichment by magnetic sorting and flow sorting. The sublibrary was amplified by PCR and cloned by homologous recombination into vectors that produce yeast-secreted scFv marked with antibody tags and a His6 tag for isolation. The scFv-secretor yeasts were arrayed and screened by capture ELISA analogous to the hybridoma screens used for mouse monoclonal antibodies. We modified the original secretion vector from PNNL, to contain an extra domain for binding to a solid surface without loss of activity. By combining antiHE4 scFvs in the original and our modified vector, we have developed a scFv-based sandwich ELISA. We are working to improve the affinity of this test and to produce assays for other potential indicators of cancer.