A functional dual-coated (FDC) microtiter plate method to replace the botulinum toxin LD50 test

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 proteas...

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Bibliographic Details
Published in:Analytical biochemistry 2012-06, Vol.425 (1), p.28-35
Main Authors: Liu, Yvonne Y.B., Rigsby, Peter, Sesardic, Dorothea, Marks, James D., Jones, Russell G.A.
Format: Article
Language:English
Subjects:
Animals
Antibodies, Monoclonal - chemistry
Antibodies, Monoclonal - metabolism
Assay
Botulinum neurotoxin
botulinum toxin
Botulinum Toxins, Type A - analysis
Botulinum Toxins, Type A - chemistry
Di-chain
disulfide bonds
Enzyme
enzyme activity
enzyme-linked immunosorbent assay
Enzyme-Linked Immunosorbent Assay - methods
epitopes
Female
Humans
Lethal Dose 50
Mice
monoclonal antibodies
phosphates
Sensitivity and Specificity
SNAP25
Therapeutic
Toxin
urea
zinc
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Description
Summary: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 (130aMLoD), 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., 1.2M 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 Q197 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.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2012.02.038