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PMID: 17113097 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Method for generation of in vivo biotinylated recombinant antibodies by yeast mating.

Journal of immunological methods ·Vol. 317 ·No. 1-2 ·2006-12-20 ·Pages 132-43

Scholler N, Garvik B, Quarles T, Jiang S, Urban N

Abstract

We describe here a novel method for generation of yeast-secreted, in vivo biotinylated recombinant antibodies, or biobodies. Biobodies are secreted by diploid yeast resulting from the fusion of two haploid yeast of opposite mating type. One yeast carries a cDNA encoding an antibody recognition sequence fused to an IgA1 hinge and a biotin acceptor site (BCCP) at the C-terminus; the other carries a cDNA encoding an E. coli biotin ligase (BirA) fused to KEX2 golgi-localization sequences, so that BirA can catalyze the biotin transfer to the recognition sequence-fused BCCP within the yeast secretory compartment. We illustrate this technology with biobodies against HE4, a biomarker for ovarian carcinoma. Anti-HE4 biobodies were derived from clones or pools of anti-HE4-specific yeast-display scFv, constituting respectively monoclonal (mBb) or polyclonal (pBb) biobodies. Anti-HE4 biobodies were secreted directly biotinylated thus bound to labeled-streptavidin and streptavidin-coated surfaces without Ni-purification. Anti-HE4 biobodies demonstrated specificity and sensitivity by ELISA assays, flow cytometry analysis and Western blots prior to any maturation; dissociation equilibrium constants as measured by surface plasmon resonance sensor were of K(d)=4.8 x 10(-9) M and K(d)=5.1 x 10(-9) M before and after Ni-purification respectively. Thus, yeast mating permits cost-effective generation of biotinylated recombinant antibodies of high affinity.

MeSH Terms
Antibodies/immunology Antibody Affinity Biotinylation Blotting, Western Cell Line Epididymal Secretory Proteins/immunology Flow Cytometry Humans Recombinant Fusion Proteins/biosynthesis,immunology Surface Plasmon Resonance Transfection Yeasts beta-Defensins
Chemicals
Antibodies DEFB126 protein, human Epididymal Secretory Proteins Recombinant Fusion Proteins beta-Defensins
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Scholler Nathalie
Translational Outcomes Research Group, Molecular Diagnostics Program, Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. nscholle@fhcrc.org
Garvik Barbara
Quarles Travis
Jiang Shaoyi
Urban Nicole
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Article Info
Journal
Journal of immunological methods
Abbr.
J Immunol Methods
ISSN
0022-1759
DOI
10.1016/j.jim.2006.10.003
Email
nscholle@fhcrc.org
Published
2006-12-20
Epub
2006-00-30
Pages
132-43
Language
English
Region
Netherlands
NLM ID
1305440
PMCID
PMC1784068
Subset
IM
Grants
NCI NIH HHS · P50 CA083636 · United States
NCI NIH HHS · P50 CA83636 · United States
External Links
PubMed source DOI: 10.1016/j.jim.2006.10.003 PMC Full Text View journal details
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