title: A functional dual-coated (FDC) microtiter plate method to replace the botulinum toxin LD50 test.
abstract: Conventional capture ("Sandwich") ELISAs equally detect denatured inactive and native active botulinum type A toxin. Light chain endoprotease activity assays also fail to distinguish between various inactive molecules including partially denatured and fragmented material still retaining this protease activity. By co-coating microtiter plates with SNAP25 substrate and a monoclonal antibody specific for a conformational epitope of the toxin's Hc domain, it was possible to develop a highly sensitive (130 aM LoD), precise (1.4% GCV) new assay specific for the biologically active toxin molecule. Capture was performed in phosphate buffer with a fixed optimal concentration of chaotropic agent (e.g., 1.2 M urea) to differentially isolate functional toxin molecules. Addition of enzymatically favorable buffer containing zinc and DTT reduced the interchain disulfide bond releasing and activating the captured L-chain with subsequent specific cleavage of the SNAP25(1-206) substrate. A neoepitope antibody specific for the newly exposed Q(197) epitope was used to quantify the cleaved SNAP25(1-197). The assay's requirement for the intact toxin molecule was demonstrated with pre-reduced toxin (heavy and light chains), recombinant LHn fragments, and stressed samples containing partially or fully denatured material. This is the first known immunobiochemical assay that correlates with in vivo potency and provides a realistic alternative.
authors.affiliation: Division of Bacteriology, National Institute for Biological Standards and Control, Health Protection Agency, South Mimms, Hertfordshire, UK.
authors.forename: Yvonne Y B
authors.initials: YY
authors.lastname: Liu
authors.forename: Peter
authors.initials: P
authors.lastname: Rigsby
authors.forename: Dorothea
authors.initials: D
authors.lastname: Sesardic
authors.forename: James D
authors.initials: JD
authors.lastname: Marks
authors.forename: Russell G A
authors.initials: RG
authors.lastname: Jones
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D019274:Botulinum Toxins, Type A / Q000032:analysis* / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D007928:Lethal Dose 50; D051379:Mice; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D019274:Botulinum Toxins, Type A