Recombinant proteins that trigger production of antibodies recognizing botulinum neurotoxin while not possessing sequences of this toxin (original) (raw)
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Botulinum neurotoxin (BoNT) serotypes A, B and E are most frequently associated with human botulism. Recombinant vaccines against BoNTs are usually based on one or more domain(s) of the toxin molecule. In this study, we investigate a new-designed multiple antigenic polypeptide for serotypes A and E (MAP/AE), containing two linear epitopes from each serotype. A synthetic gene was used to express the recombinant MAP/AE, in E. coli. Anti-MAP/AE antibodies were produced by injecting the purified MAP/AE to Balb/C mice. The interactivity of these antibodies and BoNT/A and E has been shown by ELISA. High titers of anti-MAP/AE antibodies were detected in mice sera. The anti-MAP/AE antibody titer is clearly detectable even at 25,600 dilution level. The anti-MAP/AE antibodies bound to both BoNT/A and BoNT/E holotoxin molecules. Neutralization ability of the antibodies for both toxin serotypes was determined, by an inhibitory ELISA assay. Results are suggestive of the feasibility of this epitope targeting strategy to develop novel multivalent recombinant vaccines against BoNTs.
The Journal of toxicological sciences, 2010
Botulinum neurotoxins (BoNTs) are potent toxicant proteins composed of a heavy chain (100 kDa) and a light chain (50 kDa) of seven (A-G) serotypes that is responsible for botulism syndrome. In this study, polypeptides from C-terminal heavy chain of BoNTs serotypes A, B and E to the length of 54, 45 and 48 amino acid respectively were selected, linked together using a hydrophobic linker and expressed in E. coli. The expression efficiency of the chimeric protein was found to be 51%. The chimeric protein was produced in the form of inclusion body (IB) both at two studied temperatures, 30 degrees C and 37 degrees C. This IB was extracted by ultracentrifugation and followed for chimeric protein solubilization and purification using of ultrafiltration and preparative electrophoresis. The purified chimeric protein was characterized using blotting and ELISA. To evaluate the protection ability of this chimeric antigen against their active toxins, it was injected to mice and the antibody tite...
Generation of a Recombinant Full-Length Human Antibody Binding to Botulinum Neurotoxin A
Applied Biochemistry and Biotechnology, 2010
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 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 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 (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.
Hybridoma and Hybridomics, 2003
A recombinant single-chain fragment variable antibody (scFv) to botulinum A neurotoxin (BoNT/A) was developed. BALB/C mice were immunized with BoNT/A. Splenomic RNA was isolated from the hyperimmune mice and used to prepare a cDNA library, from which the variable regions of the heavy and light chain antibody genes were generated and connected by a DNA linker. The resulting scFv genes were cloned into the phagemid vector pCANTAB5 in order to construct phage display scFv libraries. Individual anti-BoNT/A phage clones were isolated from the phage display libraries by immunoaffinity selection using immobilized BoNT/A and further evaluated by enzyme-linked immunosorbant assay, immunoprecipitation and Western blotting. Forty-eight clones were found to be BoNT/A-reactive. The most reactive clone, designated D12, was selected for further study. The scFv gene of D12 was subcloned into a Pichia pastoris vector, and expression in yeast was evaluated.
PLoS ONE, 2009
Background: Botulism, an often fatal neuroparalytic disease, is caused by botulinum neurotoxins (BoNT) which consist of a family of seven serotypes (A-H) produced by the anaerobic bacterium Clostridium botulinum. BoNT, considered the most potent biological toxin known, is a 150 kDa protein consisting of a 100 kDa heavy-chain (Hc) and a 50 kDa light-chain (Lc). F1-40 is a mouse-derived, IgG1 monoclonal antibody that binds the light chain of BoNT serotype A (BoNT/A) and is used in a sensitive immunoassay for toxin detection. We report the fine epitope mapping of F1-40 and the deduced amino acid sequence of the variable regions of the heavy and light chains of the antibody.
Hybridoma, 2011
Botulinum neurotoxins (BoNTs) are extremely potent toxins that can contaminate foods and are a public health concern. Anti-BoNT antibodies have been described that are capable of detecting BoNTs; however there still exists a need for accurate and sensitive detection capabilities for BoNTs. Herein, we describe the characterization of a panel of eight monoclonal antibodies (MAbs) generated to the non-toxic receptor-binding domain of BoNT/ A (H C 50/A) developed using a high-throughput screening approach. In two independent hybridoma fusions, two groups of four IgG MAbs were developed against recombinant H C 50/A. Of these eight, only a single MAb, F90G5-3, bound to the whole BoNT/A protein and was characterized further. The F90G5-3 MAb slightly prolonged time to death in an in vivo mouse bioassay and was mapped by pepscan to a peptide epitope in the Nterminal subdomain of H C 50/A (H CN 25/A) comprising amino acid residues 985 WTLQDTQEIKQRVVF 999 , an epitope that is highly immunoreactive in humans. Furthermore, we demonstrate that F90G5-3 binds BoNT/A with nanomolar efficiency. Together, our results indicate that F90G5-3 is of potential value as a diagnostic immunoreagent for BoNT/A capture assay development and bio-forensic analysis.
High-Affinity, Protective Antibodies to the Binding Domain of Botulinum Neurotoxin Type A
Infection and Immunity, 2001
Monoclonal antibodies (MAbs) were prepared against the putative binding domain of botulinum neurotoxin A (BoNT/A), a nontoxic 50-kDa fragment. Initially, all fusion products were screened against the holotoxin BoNT/A and against the binding fragment, BoNT/A H C . Eleven neutralizing hybridomas were cloned, and their specific binding to BoNT/A H C was demonstrated by surface plasmon resonance, with dissociation constants ranging from 0.9 to <0.06 nM. Epitope mapping by real-time surface plasmon resonance showed that the antibodies bound to at least two distinct regions of the BoNT/A H C fragment. These MAbs will be useful tools for studying BoNT/A interactions with its receptor, and they have potential diagnostic and therapeutic applications.