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

• Published in: Biotechnology and bioengineering Vol. 108; no. 10; pp. 2456 - 2467
• Main Authors: Gray, Sean A., Barr, John R., Kalb, Suzanne R., Marks, James D., Baird, Cheryl L., Cangelosi, Gerard A., Miller, Keith D., Feldhaus, Michael J.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.10.2011; Wiley; Wiley Subscription Services, Inc
• Subjects: 60 APPLIED LIFE SCIENCES; affinity reagents; ANTIBODIES; Antibodies, Bacterial - biosynthesis; Antibodies, Bacterial - chemistry; Antibodies, Bacterial - genetics; Antibody Specificity; ANTIGENS; Binding; Binding sites; Biological and medical sciences; Biotechnology; BoNT/A; Botulinum Toxins, Type A - chemistry; Botulinum Toxins, Type A - genetics; Botulinum Toxins, Type A - metabolism; CHAINS; CLEAVAGE; Cloning; CLOSTRIDIUM BOTULINUM; DETECTION; EFFICIENCY; Epitopes - chemistry; Epitopes - genetics; Epitopes - metabolism; Fundamental and applied biological sciences. Psychology; Humans; INCUBATION; Marking; MASS SPECTROSCOPY; molecular probes; MONOCLONAL ANTIBODIES; PEPTIDES; PRODUCTION; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; scFv; Single-Chain Antibodies - biosynthesis; Single-Chain Antibodies - chemistry; Single-Chain Antibodies - genetics; TOXINS; Yeast; yeast display; YEASTS; Clostridium botulinum; Yeast; Antigenic determinant; BoNT/A; Selection; Clostridiales; Metalloendopeptidases; Fv-Fragment; Synergism; Bontoxilysin; affinity reagents; Bacteria; Molecular probe; antibodies; Enzyme; Antibody; yeast display; Peptidases; Toxin; Screening; scFv; Clostridiaceae; Neurotoxin; Hydrolases; Affinity; molecular probes
• ISSN: 0006-3592; 1097-0290; 1097-0290
• DOI: 10.1002/bit.23196

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, 3 also bound to full‐length BoNT/A toxin complex with affinities ranging from 5 to 48 nM. Epitope binning showed that the three unique clones recognized at least two epitopes distinct from one another as well as from the detection MAbs. After production in E. coli, 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 antigens. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support. Biotechnol. Bioeng. 2011;108: 2456–2467. © 2011 Wiley Periodicals, Inc.