title: Rapid microplate assay for monitoring botulinum neurotoxin B catalytic activity.
abstract: The binding activity of a rabbit polyclonal antiserum raised against a 51-residue peptide (P51) homologous to human VAMP2 (residues 44-94) was examined. Human VAMP2 is an 18-kDa protein located on the external membrane of small synaptic vesicles and is targeted by four of the seven botulinum neurotoxin (BoNT) serotypes (B, D, F and G). The antiserum, designated anti-P51, recognized P51 but exhibited little cross-reactivity with the two cleavage products that result from BoNT/B-mediated proteolysis of P51. The larger of these fragments, designated as P33 (residues 44-76), exhibited a weak but measurable interaction with the antiserum. The smaller cleavage product, designated as P18 (residues 77-94), was not recognized by the antiserum. Anti-P51 was used to monitor BoNT/B light chain (LC)-mediated cleavage of P51 using an indirect ELISA. The serine protease inhibitor phenylmethylsulfonyl fluoride did not inhibit BoNT/B activity, but the zinc chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) and the elastase inhibitor 7- N -phenylcarbamoylamino-4-chloro-3-propyloxyisocoumarin (ICD 1578) produced complete blockade of BoNT/B LC action. Under ideal conditions, it will be possible to evaluate up to seven candidate anti-BoNT/B drugs in triplicate at four concentrations using a single 96-well microtiter plate. These findings indicate that the ELISA will be suitable for rapid screening of BoNT/B inhibitors.
authors.affiliation: Neurotoxicology Branch, Pharmacology Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Keller
authors.forename: J L
authors.initials: JL
authors.lastname: Nowakowski
authors.forename: M G
authors.initials: MG
authors.lastname: Filbert
authors.forename: M
authors.initials: M
authors.lastname: Adler
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019274:Botulinum Toxins, Type A; D002384:Catalysis; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D050683:R-SNARE Proteins; D011817:Rabbits
chemical_list: D008565:Membrane Proteins; D011480:Protease Inhibitors; D050683:R-SNARE Proteins; C096323:rimabotulinumtoxinB; D008666:Metalloendopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A