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Ultra Sensitive HPLC Detection Assay for Botulinum Neurotoxin Type A

Christian, T.; Suryadi, K.; Shine, N.

The botulinumneurotoxins are among the most potent toxins in nature. They are synthesized as single 150 kDapolypeptide chains which are subsequently cleaved to produce a 100 kDaheavy chain and a 50 kDa light chain linked by a disulfide bond. Four of the seven immunologically distinct serotypes, A, B, E, and F, cause botulism in humans. The 50 kDalight chain of each serotype is a zinc endoproteasethat cleaves a single target protein which is essential for synaptic vesicle membrane fusion. This inhibits neurotransmitter release which leads to muscular paralysis. These secreted toxins are regarded as major biological warfare threats. Due to their extreme potency and lethality, detection of these toxins requires a highly sensitive and reliable assay. Measurement of proteolytic activity provides a potentially sensitive and direct means for detecting these potent toxins. Substantial signal amplification can be expected as a result of catalytic turnover of the substrate. Each serotype of the neurotoxin selectively cleaves one membrane fusion protein at a specific site, creating an unique cleaved product, providing the basis for an inherently specific assay. AsensitiveassayfocusedontheenzymaticpropertyofbotulinumtoxintypeA(BoNT/A)hasbeen developed.Thesensitivityissignificantlyimprovedbytheuseofhighaffinitymonoclonalantibodies,one specificfortheheavychainandoneforthelightchain,tocaptureandconcentratethetoxinpriortoaddition ofthesubstrate.TheFRETpeptideSNAPtide,designedatListBiologicalLaboratories,wasusedasthe substrateforBoNT/A.GenerationofcleavedpeptidewasmonitoredusingreversephaseHPLCwitha fluorescencedetector.Inaninitialstudy,cleavageof20MSNAPtidepeptidesubstrateby5pg/ml BoNT/Aatroomtemperaturewasdetectedinanovernightdigestion.Thisstudydemonstratesthatthis methodcanbeusedtoachievetheprerequisitesensitivityforBoNT/Awhichis20pg/ml,comparabletothe amountdetectedusingthegoldstandardmousebioassay.