Substrates and Controls for the Quantitative Detection of Active Botulinum Neurotoxin in Protease-Containing Samples
作者:Karine Bagramyan、Bruce E. Kaplan、Luisa W. Cheng、Jasmin Strotmeier、Andreas Rummel、Markus Kalkum
DOI:10.1021/ac4008418
日期:2013.6.4
Botulinum neurotoxins (BoNTs) are used in a wide variety of medical applications, but there is limited pharmacokinetic data on active BoNT. Monitoring BoNT activity in the circulation is challenging because BoNTs are highly toxic and are rapidly taken up by neurons and removed from the bloodstream. Previously we reported a sensitive BoNT “Assay with a Large Immunosorbent Surface Area” that uses conversion of fluorogenic peptide substrates to measure the intrinsic endopeptidase activity of bead-captured BoNT. However, in complex biological samples, protease contaminants can also cleave the substrates, reducing sensitivity and specificity of the assay. Here, we present a novel set of fluorogenic peptides that serve as BoNT-specific substrates and protease-sensitive controls. BoNT-cleavable substrates contain a C-terminal Nle, while BoNT-noncleavable controls contain its isomer ε-Ahx. The substrates are cleaved by BoNT subtypes A1-A3 and A5. Substrates and control peptides can be cleaved by non-BoNT proteases (e.g., trypsin, proteinase K, and thermolysin) while obeying Michaelis–Menten kinetics. Using this novel substrate/control set, we studied BoNT/A1 activity in two mouse models of botulism. We detected BoNT/A serum activities ranging from ∼3600 to 10 amol/L in blood of mice that had been intravenously injected 1 h prior with BoNT/A1 complex (100 to 4 pg/mouse). We also detected the endopeptidase activity of orally administered BoNT/A1 complex (1 μg) in blood 5 h after administration; activity was greatest 7 h after administration. Redistribution and elevation rates for active toxin were measured and are comparable to those reported for inactive toxin.
肉毒杆菌神经毒素(BoNTs)被广泛应用于各种医疗领域,但有关活性 BoNT 的药代动力学数据却十分有限。监测血液循环中的 BoNT 活性具有挑战性,因为 BoNT 毒性很强,会迅速被神经元吸收并从血液中排出。我们以前曾报道过一种灵敏的 BoNT "大免疫吸附表面积测定法",该方法利用含氟肽底物的转化来测量捕获珠的 BoNT 的内在内肽酶活性。然而,在复杂的生物样本中,蛋白酶污染物也会裂解底物,从而降低检测的灵敏度和特异性。在此,我们提出了一组新型的荧光肽,可作为 BoNT 特异性底物和蛋白酶敏感性对照。BoNT可裂解底物含有一个C端Nle,而BoNT不可裂解对照组含有其异构体ε-Ahx。底物由 BoNT 亚型 A1-A3 和 A5 裂解。底物和对照肽可被非 BoNT 蛋白酶(如胰蛋白酶、蛋白酶 K 和热溶解酶)裂解,同时遵循 Michaelis-Menten 动力学。利用这种新型底物/对照组,我们在两种肉毒中毒小鼠模型中研究了 BoNT/A1 的活性。我们在 1 小时前静脉注射 BoNT/A1 复合物(100 至 4 pg/只小鼠)的小鼠血液中检测到了从 3600 至 10 amol/L 不等的 BoNT/A 血清活性。我们还检测了口服 BoNT/A1 复合物(1 μg)5 小时后血液中的内肽酶活性;给药 7 小时后活性最高。对活性毒素的再分布和升高率进行了测定,其结果与所报道的非活性毒素的再分布和升高率相当。