Centre d'Analyse Protéomique de Marseille (CAPM), Faculté de Médecine secteur nord, Université de la Méditerranée (IFR 11), Marseille F-13916, France.
Anal Biochem. 2011 Mar 15;410(2):281-8. doi: 10.1016/j.ab.2010.11.045. Epub 2010 Dec 4.
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.
肉毒神经毒素（BoNTs）是已知的最毒物质之一。监测和诊断需要在食品和人血清等复杂介质中快速检测 BoNTs 的方法。我们开发了体外测定法，可在数分钟的时间尺度内特异性检测肉毒神经毒素 B（BoNT/B）的蛋白酶活性。使用实时表面等离子体共振监测 BoNT/B 底物 VAMP2 的切割，该底物是与突触小泡相关的膜 SNARE 蛋白，通过特异性抗体与微芯片偶联来测量囊泡的捕获。该测定法在低离子强度缓冲液中起作用，并且在很宽的 pH 值范围内（5.5-9.0）稳定。用 2 pM 天然 BoNT/B 全毒素在 10 分钟内检测到 VAMP2 的内切蛋白酶切割。在 3 小时内，发现胡萝卜汁、苹果汁和牛奶等液体食品产品中存在低皮摩尔量的 BoNT/B 污染。在 B 型肉毒中毒患者的血清中检测到 BoNT/B 活性，但在健康对照者或其他神经疾病患者的血清中未检测到。这种稳健、灵敏、快速的蛋白质芯片测定法适用于监测食品产品中的 BoNT/B，也可用于 B 型肉毒中毒的诊断测试，并且可以替代当前的体内小鼠生物测定法。
