Selection and characterization of a human monoclonal neutralizing antibody for Clostridium Botulinum neurotoxin serotype B

Zhou, Helin; Zhou, Bin; Pellett, Sabine; Johnson, Eric A.; Janda, Kim D.

doi:10.1016/j.bmcl.2008.12.055

Cited by 10 publications

(10 citation statements)

References 21 publications

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“…This is a first kind of report which shows that the antibody raised against the light chain are able to prevent the blockage of acetylcholine release by in-vitro studies using PC-12 neuronal cells. Similar kind of studies were reported in BoNT/B HC domain (1088-1295) as antigen to screen BoNT/B neutralizing potency by a sensitive in-vitro cell-based assay with primary rat spinal cord (RSC) cells and the results indicates that a 1,000-fold molar excess of the Fab F2 was required for inhibition of BoNT/B activity and it concluded that this Fab possesses weak neutralization activity [38].…”

Section: Estimation Of Acetylcholine Releasementioning

confidence: 62%

Expression, Purification and Development of Neutralizing Antibodies from Synthetic BoNT/B LC and Its Application in Detection of Botulinum Toxin Serotype B

Ponmariappan

Jain²,

Sijoria³

et al. 2012

PPL

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Botulism is a neuroparalytic disease caused by Clostridium botulinum, which produces seven (A-G) neurotoxins (BoNTs). The mouse bioassay is the gold standard for the detection of botulinum neurotoxins, however it requires at least 3-4 days for completion. Most of the studies were carried out in botulinum toxin A and less on type B. Attempts have been made to develop an ELISA based detection system, which is potentially an easier and more rapid method of botulinum neurotoxin detection. In the present study, the synthetic BoNT/B LC gene was constructed using PCR overlapping primers, cloned in a pET28a+ vector and expressed in E. coli BL21DE3. The maximum yield of recombinant proteins was optimized after 16 hrs of post induction at 21°C and purified the recombinant protein in soluble form. Antibodies were raised in Mice and Rabbit. The IgG antibody titer in the case of Mice was 1: 1,024,000 and Rabbit was 1: 512,000 with alum as adjuvant via intramascular route. The biological activity of the recombinant protein was confirmed by in-vitro studies using PC12 cells by the synaptobrevin cleavage, the rBoNT/B LC protein showed the maximum blockage of acetylcholine release at a concentration of 150nM rBoNT/B LC in comparison to the control cells. When the cells were incubated with rBoNT/B LC neutralized by the antisera raised against it, the acetylcholine release was equivalent to the control. IgG specific to rBoNT/B LC was purified from raised antibodies. The results showed that the developed antibody against rBoNT/B LC protein were able to detect botulinum toxin type B approximately up to 1 ng/ml. These developed high titer antibodies may prove useful for the detection of botulinum neurotoxins in food and clinical samples.

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Section: Estimation Of Acetylcholine Releasementioning

confidence: 62%

Expression, Purification and Development of Neutralizing Antibodies from Synthetic BoNT/B LC and Its Application in Detection of Botulinum Toxin Serotype B

Ponmariappan

Jain²,

Sijoria³

et al. 2012

PPL

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“…The 50-kDa C-terminal fragment of the HC, known as the binding domain, recognizes surface receptors on target neurons, thus provoking the transmembrane internalization of the whole toxin [4,5]. Botulism can be prevented by the presence of neutralizing antibodies (or vaccination) against the botulinum neurotoxins [1]. The current formalin-inactivated pentavalent vaccine for botulism is effective, but this vaccine requires large scale production of the highly toxic material, which involves extensive treatment with formalin [6].…”

Section: Introductionmentioning

confidence: 99%

“…The botulinum neurotoxins (BoNTs), synthesized by a family of seven toxinotypes (A-G), are responsible for potent food intoxication known as botulism, which is a severe neuroparalytic disease [1]. BoNTs possess similar structures but are immunologically distinct, each of which is expressed as the single polypeptide chain of 150 kDa [2].…”

Section: Introductionmentioning

confidence: 99%

High level expression of recombinant BoNT/A-Hc by high cell density cultivation of Escherichia coli

Yari

Fatemi

Tavallaei

2011

Bioprocess Biosyst Eng

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The carboxylic domain of the Clostridium botulinum neurotoxin heavy chain (BoNT/A-HC), which has been reported as a vaccine candidate, contains the principle protective antigenic determinants. In this study, the high level expression of the BoNT/A-Hc was achieved by high cell density cultivation of recombinant Escherichia coli in a 2-l batch stirred-tank bioreactor. In order to maximize protein expression, post-induction time and IPTG inducer concentration were optimized by the Taguchi statistical design method. Results showed that the middle of the logarithmic phase and an IPTG concentration of 1 mM presented the optimum conditions for the maximum expression of BoNT/A-HC. High cell density cultivation was subsequently carried out as an effective strategy for the high level expression of recombinant BoNT/A-Hc. Consequently, soluble BoNT/A-Hc was produced at the maximum level of 486 mg l(-1), at 3 h post-induction, which was approximately 9.3 and 7.8 times higher than the levels produced by the shake flask and batch culturing methods, respectively.

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“…Effective treatment for botulism involves the timely administration of neutralizing antitoxin and several commercial preparations are available for treatment of humans [ 8 ], including infant botulism [ 9 ]. New countermeasures in development include human or humanized recombinant monoclonal antibodies [ 10 , 11 , 12 , 13 ]. The traditional mouse lethality or systemic toxicity neutralization test for determination of the potency of therapeutic antitoxins has been considered at the forefront for replacement on ethical grounds [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E

Bak

Rajagopal

Stickings

et al. 2017

Toxins

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Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay.

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Selection and characterization of a human monoclonal neutralizing antibody for Clostridium Botulinum neurotoxin serotype B

Cited by 10 publications

References 21 publications

Expression, Purification and Development of Neutralizing Antibodies from Synthetic BoNT/B LC and Its Application in Detection of Botulinum Toxin Serotype B

Expression, Purification and Development of Neutralizing Antibodies from Synthetic BoNT/B LC and Its Application in Detection of Botulinum Toxin Serotype B

High level expression of recombinant BoNT/A-Hc by high cell density cultivation of Escherichia coli

SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E

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