Flow cytometry-based methods for assessing soluble scFv activities and detecting pathogen antigens in solution

Journal Article · Thu Apr 01 04:00:00 EDT 2010 · Biotechnology and Bioengineering, 105(5):973-981

OSTI ID:981998

Gray, Sean; Weigel, Kris M; Miller, Keith D; Ndung'u, Joseph; Buscher, Philippe; Tran, Thao N; Baird, Cheryl L; Cangelosi, Gerard A

Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast-display libraries. Yeast-display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from nonimmune libraries is the conversion of highly active yeast-displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast-display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeastdisplayed and secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of its ability to competitively inhibit the binding of biotinylated antigen to yeast-displayed scFv. The second is an epitope binning assay that uses secreted scFv toidentify additional yeast-displayed scFv that bind nonoverlapping or noncompeting epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast-displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast-display libraries.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (US)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 981998
• Report Number(s): PNNL-SA-68697
• Journal Information: Biotechnology and Bioengineering, 105(5):973-981, Journal Name: Biotechnology and Bioengineering, 105(5):973-981 Journal Issue: 5 Vol. 105; ISSN BIBIAU; ISSN 0006-3592
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
99 GENERAL AND MISCELLANEOUS
ANTIBODIES
ANTIGENS
BYPASSES
CHAINS
CONVERSION
GLYCOPROTEINS
IMPLEMENTATION
INHIBITION
LIBRARIES
PATHOGENS
PURIFICATION
REAGENTS
SECRETION
SOLUBILITY
SOLUTIONS
STANDARDS
SURFACES
TRYPANOSOMA
USES
YEASTS
affinity reagents
antibody
biochemistry
pathogens
scFv