Synergistic capture of Clostridium botulinum Type A neurotoxin by scFv antibodies to novel epitopes

Journal Article · Sat Oct 01 04:00:00 EDT 2011 · Biotechnology and Bioengineering, 108(10):2456-2467

DOI:https://doi.org/10.1002/bit.23196· OSTI ID:1025657

Gray, Sean A; Barr, John R; Kalb, Suzanne R; Marks, James D; Baird, Cheryl L; Cangelosi, Gerard A; Miller, Keith D; Feldhaus, Michael J

A non-immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (Hc)] with the goal of identifying scFv to novel epitopes. To do this, an antibody-mediated labeling strategy was used in which antigen-binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, three also bound to full-length BoNT/A toxin complex with affinities ranging from 5 nM to 170 nM. Epitope binning showed that the three unique clones recognized at least two epitopes that were distinct from one another and from the detection MAbs. After production in E. coli, the scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep-MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A-specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep-MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep-MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigen. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (US)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 1025657
• Report Number(s): PNNL-SA-78019
• Journal Information: Biotechnology and Bioengineering, 108(10):2456-2467, Journal Name: Biotechnology and Bioengineering, 108(10):2456-2467 Journal Issue: 10 Vol. 108; ISSN BIBIAU; ISSN 0006-3592
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
ANTIBODIES
ANTIGENS
CHAINS
CLEAVAGE
CLOSTRIDIUM BOTULINUM
DETECTION
EFFICIENCY
INCUBATION
MASS SPECTROSCOPY
MONOCLONAL ANTIBODIES
PEPTIDES
PRODUCTION
SACCHAROMYCES CEREVISIAE
TOXINS
YEASTS