A protein chip membrane-capture assay for botulinum neurotoxin activity.
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(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(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.
Institut National de la Santé et de la Recherche Médicale, Unité 641, Marseille F-13916, France.
Séverine
S
Marconi
Géraldine
G
Ferracci
Maëlys
M
Berthomieu
Shunji
S
Kozaki
Raymond
R
Miquelis
José
J
Boucraut
Michael
M
Seagar
Christian
C
Lévêque
D000818:Animals; D001905:Botulinum Toxins / Q000633:toxicity*; D019274:Botulinum Toxins, Type A / Q000633:toxicity*; D001921:Brain / Q000166:cytology; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D040081:Protein Array Analysis; D051381:Rats; D020349:Surface Plasmon Resonance; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism*
C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A