Rapid microplate assay for monitoring botulinum neurotoxin B catalytic activity.

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.