A label-free biosensor assay for botulinum neurotoxin B in food and human serum.
Botulinum neurotoxins (BoNTs) are among the most toxic substances known. Surveillance and diagnostics require methods for rapid detection of BoNTs in complex media such as foodstuffs and human serum. We have developed in vitro assays to specifically detect the protease activity of botulinum neurotoxin B (BoNT/B) on a time scale of minutes. Cleavage of the BoNT/B substrate VAMP2, a membrane SNARE protein associated with synaptic vesicles, was monitored using real-time surface plasmon resonance to measure vesicle capture by specific antibodies coupled to microchips. The assay is functional in low-ionic-strength buffers and stable over a wide range of pH values (5.5-9.0). Endoproteolytic cleavage of VAMP2 was detected in 10 min with 2 pM native BoNT/B holotoxin. Contamination of liquid food products such as carrot juice, apple juice, and milk with low picomolar amounts of BoNT/B was revealed within 3h. BoNT/B activity was detected in sera from patients with type B botulism but not in healthy controls or patients with other neurological diseases. This robust, sensitive, and rapid protein chip assay is appropriate for monitoring BoNT/B in food products and diagnostic tests for type B botulism and could replace the current in vivo mouse bioassay.
Centre d'Analyse Protéomique de Marseille (CAPM), Faculté de Médecine secteur nord, Université de la Méditerranée (IFR 11), Marseille F-13916, France.
Géraldine
G
Ferracci
Séverine
S
Marconi
Christelle
C
Mazuet
Emmanuel
E
Jover
Marie-Pierre
MP
Blanchard
Michael
M
Seagar
Michel
M
Popoff
Christian
C
Lévêque
D000818:Animals; D001681:Biological Assay / Q000379:methods; D015374:Biosensing Techniques / Q000379:methods*; D001905:Botulinum Toxins / Q000032:analysis* / Q000097:blood / Q000737:chemistry*; D019274:Botulinum Toxins, Type A; D001906:Botulism / Q000097:blood / Q000175:diagnosis; D003014:Clostridium botulinum / Q000201:enzymology; D005502:Food; D005504:Food Analysis / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D040081:Protein Array Analysis / Q000379:methods; D051381:Rats; D044967:Serum; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance / Q000379:methods; D013572:Synaptic Vesicles / Q000737:chemistry / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000737:chemistry* / Q000378:metabolism*
D050988:Vesicle-Associated Membrane Protein 2; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A