Generation of a Recombinant Full-Length Human Antibody Binding to Botulinum Neurotoxin A

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Authors
  1. Hu, W-G.
  2. Jager, S.
  3. Chau, D.
  4. Mah, D.
  5. Nagata, L.P.
Corporate Authors
Defence R&D Canada - Suffield, Ralston ALTA (CAN)
Abstract
In order to develop a recombinant full-length human anti-botulinum neurotoxin A (BoNT/A) antibody, human peripheral blood mononuclear cells (PBMC) were collected from three healthy volunteers and induced for BoNT/A-specific immune response by in vitro immunization. The genes encoding human Fd fragment, consisting of antibody heavy chain variable region and constant region 1 with the genes encoding antibody light chain, were cloned from the immunized PBMC. Afterwards, one combinatory human antigenbinding fragment (Fab) library was constructed using a lambda phage vector system. The size of the constructed library was approximately 105 Escherichia coli transformants. After screening the library by BoNT/A antigen using a plaque lifting with immunostaining approach, 55 clones were identified as positive. The Fab gene of the most reactive clone exhibiting particularly strong BoNT/A binding signal was further subcloned into a fulllength human IgG1 antibody gene template in an adenoviral expression vector, in which the heavy and light chains were linked by a foot-and-mouth-disease virus-derived 2A selfcleavage peptide under a single promoter. After the full-length human IgG1 was expressed in mammalian cells and purified with protein L column, sodium dodecyl sulfatepolyacrylamide gel electrophoresis showed that the heavy and light chains of the antibody were cleaved completely. The affinity expressed as the dissociation constant (Kd) for the recombinant human antibody to b
Report Number
DRDC-SUFFIELD-SL-2008-217 — Scientific Literature
Date of publication
01 Dec 2008
Number of Pages
11
DSTKIM No
CA033667
CANDIS No
533102
Format(s):
Electronic Document(PDF)

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