A protein chip membrane-capture assay for botulinum neurotoxin activity

Journal Article · Mon Dec 15 04:00:00 EST 2008 · Toxicology and Applied Pharmacology

DOI:https://doi.org/10.1016/j.taap.2008.09.005· OSTI ID:21180511

Marconi, Severine ^[1]; Ferracci, Geraldine ^[2]; Berthomieu, Maelys ^[1]; Kozaki, Shunji ^[3]; Miquelis, Raymond ^[4]; Boucraut, Jose ^[4]; Seagar, Michael ^[1]

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1. Institut National de la Sante et de la Recherche Medicale, Unite 641, Marseille F-13916 (France)
2. Centre d'Analyse Proteomique de Marseille (CAPM), Institut Jean Roche (IFR 11), Faculte de medecine secteur nord, Bd P. Dramard, Marseille F-13916 (France)
3. Department of Veterinary Sciences, Osaka prefecture University, 1-1 Gakuen-cho, Sakai-shi, Osaka 599-8531 (Japan)
4. Universite de la Mediterranee-Aix Marseille 2, Faculte de medecine secteur nord, Bd P. Dramard, Marseille F-13916 (France)

Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC{sub 50}s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC{sub 50} of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays.

• OSTI ID: 21180511
• Journal Information: Toxicology and Applied Pharmacology, Journal Name: Toxicology and Applied Pharmacology Journal Issue: 3 Vol. 233; ISSN TXAPA9; ISSN 0041-008X
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
BRAIN
DRUGS
ENZYME IMMUNOASSAY
FLUORESCENCE
IN VITRO
IN VIVO
MEMBRANE PROTEINS
MEMBRANES
MICE
MONOCLONAL ANTIBODIES
NERVE CELLS
PATHOGENS
RESONANCE
SUBSTRATES
TOXINS