A new peptide substrate for enhanced botulinum neurotoxin type B detection by endopeptidase–liquid chromatography–tandem mass spectrometry/multiple reaction monitoring assay

Rosen, Osnat; Feldberg, Liron; Gura, Sigalit; Zichel, Ran

doi:10.1016/j.ab.2014.09.016

Cited by 17 publications

(12 citation statements)

References 28 publications

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“…We tested the feasibility of multiplex analysis of BoNT/A, B and E using our newly designed BoNT/A substrate together with the previously reported BoNT/B and E substrates 14 , 15 . To this end, the three substrates for the three BoNT serotypes were mixed in a single reaction.…”

Section: Resultsmentioning

confidence: 99%

Development of a multiplex Endopep-MS assay for simultaneous detection of botulinum toxins A, B and E

Rosen

Feldberg

Yamin

et al. 2017

Sci Rep

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Botulinum neurotoxins (BoNTs) are bacterial proteins that cause botulism, a life-threatening disease. The Endopep-MS assay permits rapid detection and serotypic differential diagnosis of BoNTs. The serotype-specific nature of this assay requires that each serum sample be aliquoted and individually tested, which in addition to the limited volume of clinical samples, especially in infants, points to the need for a multiplex assay. However, previous attempts to develop such an assay have been challenging, mainly due to inhibition of BoNT/A activity by the BoNT/E peptide substrate. BoNT/A and BoNT/E share the same native target protein as their substrate. We hypothesized that the steric interference between the BoNT/A and BoNT/E substrate peptides is responsible for the difficulty in simultaneously assaying these two toxins. To explore the basis for steric interference, we used the reported structure of BoNT/A in complex with SNAP-25 and modelled the structure of BoNT/E with SNAP-25. Following this thorough structural analysis, we designed a new peptide substrate for BoNT/A that maintained the assay sensitivity and allowed, for the first time, simultaneous detection of the three most abundant human botulinum serotypes. Adopting the multiplex assay will minimize the required sample volume and assay time for botulinum detection while maintaining the superior Endopep-MS assay performance.

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Section: Resultsmentioning

confidence: 99%

Development of a multiplex Endopep-MS assay for simultaneous detection of botulinum toxins A, B and E

Rosen

Feldberg

Yamin

et al. 2017

Sci Rep

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“…Endo-peptidase activity was measured as previously described [51,52,53,54,55]. Briefly, the reaction was performed in a 20-µL reaction volume containing toxin-bound beads, 100 µM peptide substrate, 1 mM ZnCl 2 , 1% Triton, 10 mM dithiothreitol (DTT), and 50 mM HEPES buffer (pH 7.3) at 37 °C with shaking for 5 h. One hundred eighty microliters of 1% formic acid was added, and the samples were then incubated for 2 min at 100 °C.…”

Section: Methodsmentioning

confidence: 99%

Role of Homologous Fc Fragment in the Potency and Efficacy of Anti‐Botulinum Antibody Preparations

Torgeman

Ozeri

David

et al. 2017

Toxins

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The only approved treatment for botulism relies on passive immunity which is mostly based on antibody preparations collected from hyper-immune horses. The IgG Fc fragment is commonly removed from these heterologous preparations to reduce the incidence of hyper-sensitivity reactions. New-generation therapies entering the pipeline are based on a combination of humanized monoclonal antibodies (MAbs), which exhibit improved safety and pharmacokinetics. In the current study, a systematic and quantitative approach was applied to measure the direct contribution of homologous Fc to the potency of monoclonal and polyclonal antitoxin preparations in mice. Homologous Fc increased the potency of three individual anti-botulinum toxin MAbs by up to one order of magnitude. Moreover, Fc fragment removal almost completely abolished the synergistic potency obtained from a combined preparation of these three MAbs. The MAb mixture neutralized a 400-mouse median lethal dose (MsLD50) of botulinum toxin, whereas the F(ab′)2 combination failed to neutralize 10 MsLD50 of botulinum toxin. Notably, increased avidity did not compensate for this phenomenon, as a polyclonal, hyper-immune, homologous preparation lost 90% of its potency as well upon Fc removal. Finally, the addition of homologous Fc arms to a heterologous pharmaceutical anti-botulinum toxin polyclonal horse F(ab′)2 preparation improved its efficacy when administered to intoxicated symptomatic mice. Our study extends the aspects by which switching from animal-based to human-based antitoxins will improve not only the safety but also the potency and efficacy of passive immunity against toxins.

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“…Endo-peptidase activity was measured as described previously [18,29,41,42,43]. Briefly, the reaction was performed in a 20 µL reaction volume containing toxin-bound beads, 100 µM peptide substrate, 1 mM ZnCl 2 , 1% Triton, 10 mM dithiothreitol (DTT), and 50 mM HEPES buffer (pH 7.3) at 37 °C with shaking for five hours.…”

Section: Methodsmentioning

confidence: 99%

Development of an Innovative in Vitro Potency Assay for Anti-Botulinum Antitoxins

Rosen

Ozeri

Barnea

et al. 2016

Toxins

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Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming, labor-intensive, costly, and raises ethical issues related to the large numbers of laboratory animals needed. Until now, all efforts to develop an alternative in vitro assay have not provided a valid replacement to the mouse potency assay. In the present study, we report the development of an innovative in vitro assay for determining botulinum antitoxin potency, using botulinum type B as a model. The concept of the assay is to mimic two fundamental steps in botulinum intoxication: receptor binding and catalytic activity. By simulating these steps in vitro we were able to accurately determine the potency of antitoxin preparations. The reproducibility of the assay was high with a CV < 13%. Most importantly, the antitoxin potency measured by the in vitro assay highly correlated with that measured by the standard in vivo mouse assay (r = 0.9842, p < 0.0001). Thus, this new in vitro assay has the potential to be considered, after validation, as a replacement to the mouse assay for quantitating neutralizing antibody concentrations in pharmaceutical botulinum antitoxin preparations. Future adoption of this in vitro assay would minimize the use of laboratory animals, speed up the time, and reduce the cost of botulinum antitoxin approval.

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A new peptide substrate for enhanced botulinum neurotoxin type B detection by endopeptidase–liquid chromatography–tandem mass spectrometry/multiple reaction monitoring assay

Cited by 17 publications

References 28 publications

Development of a multiplex Endopep-MS assay for simultaneous detection of botulinum toxins A, B and E

Development of a multiplex Endopep-MS assay for simultaneous detection of botulinum toxins A, B and E

Role of Homologous Fc Fragment in the Potency and Efficacy of Anti‐Botulinum Antibody Preparations

Development of an Innovative in Vitro Potency Assay for Anti-Botulinum Antitoxins

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