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

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 antigen-binding fragment (Fab) library was constructed using a lambda phage vector system. The size of the constructed library was approximately 10(5) 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 full-length 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 self-cleavage peptide under a single promoter. After the full-length human IgG1 was expressed in mammalian cells and purified with protein L column, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the heavy and light chains of the antibody were cleaved completely. The affinity expressed as the dissociation constant (K(d)) for the recombinant human antibody to bind to BoNT/A was determined by indirect enzyme-linked immunosorbent assay and results confirmed that the recombinant full-length human antibody retained BoNT/A-binding specificity with K(d) value of 10(-7) M.

Humanization and Characterization of an Anti-Ricin Neutralization Monoclonal Antibody

“…A recombinant adenovirus vector, pAdhD9 expressing hD9 was constructed and the recombinant hD9 was expressed in HEK 293 mammalian cells using the AdEasy system (Qbiogene, Carlsbad, CA) [29], [30]. The expressed recombinant hD9 was purified using ImmunoPure Protein (L) agarose gel (Pierce, Brockville, ON).…”

Antibody Fragments and Their Purification by Protein L Affinity Chromatography

“…Such affinity resins have been used in the noncommercial purification of scFv's [31,41,80,89,105,106], Fab's [91,[106][107][108], and single-domain antibodies [52,91,98,103,108,109].…”

Generation of Recombinant Antibodies Against Toxins and Viruses by Phage Display for Diagnostics and Therapy

“…So far, antibodyphage display was successfully used for antibody selection against a panel of toxins classified as category A agents, such as from Clostridium botulinum (botulism) [16,25,53,79] and Bacillus anthracis (anthrax) [97] and also against different category B agents, such as staphylococcal enterotoxin B [66,110] and ricin toxin from Ricinus communis [6,96]. An example for a high-risk microorganism that produces the most toxic substances known with the highest risk of potential use as bioweapons is the Gram-positive, anaerobic, spore-forming bacterium Clostridium botulinum and other Clostridium subspecies.…”

“…Phage display technology was also used for isolation of single domain antibodies (VHH) after immunization of a llama with a cocktail of seven BoNT toxoids (A-F) [25]. Another approach was the generation of a human antibody gene library after inducing a BoNT/A-specific immune response by in vitro immunization [53]. Furthermore, antibody phage display was used to generate antibodies against other clostridial toxins such as from Clostridium tetani or Clostridium difficile [17,50].…”