Synaptic vesicle chips to assay botulinum neurotoxins.
BoNTs (botulinum neurotoxins), considered to be the most toxic of all biological substances, inhibit neurotransmission through proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins [VAMP (vesicle-associated membrane protein, or synaptobrevin), SNAP-25 (25 kDa synaptosome-associated protein) or syntaxin]. Expansion in the use of BoNTs as therapeutic and cosmetic agents, and the potential threat they constitute as biological weapons, underlines the need for rapid and sensitive in vitro assays. Here, we present new automatized bioassays to detect VAMP cleavage by BoNT/B and F. Western blotting and SPR (surface plasmon resonance) methods revealed that BoNT/B and F totally cleave their substrate on immunoisolated SVs (synaptic vesicles). Real-time monitoring of the immunocapture of native SVs from crude lysates on SPR sensor chips enabled the detection of picogram amounts of different SV proteins. Pre-incubation of a membrane fraction containing SVs with BoNT specifically inhibited capture by anti-VAMP antibodies, and amounts as low as 0.1 pg of BoNT/B were detected. This automated SPR assay is approx. 200 times more sensitive, and 25 times more rapid, than the in vivo BoNT/B test currently used. Moreover, the method can be performed using a few thousand cultured neurons and constitutes a new screening assay for inhibitors. Our data indicate that native VAMP is an optimal substrate for in vitro BoNT assays that can be monitored by SPR.
Ferracci
Géraldine
G
Unité de Méthodologie des Interactions Moléculaires, Institut Jean Roche, Faculté de Médecine secteur Nord, 13916 Marseille, France.
Miquelis
Raymond
R
Kozaki
Shunji
S
Seagar
Michael
M
Lévêque
Christian
C
D000818:Animals; D001331:Automation; D001681:Biological Assay / Q000295:instrumentation* / Q000379:methods*; D001905:Botulinum Toxins / Q000032:analysis*; D002457:Cell Extracts / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D009474:Neurons / Q000737:chemistry; D051381:Rats; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013572:Synaptic Vesicles / Q000378:metabolism*; D013997:Time Factors; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism
D002457:Cell Extracts; D050988:Vesicle-Associated Membrane Protein 2; D001905:Botulinum Toxins