Rapid microplate assay for monitoring botulinum neurotoxin B catalytic activity

The binding activity of a rabbit polyclonal antiserum raised against a 51-r esidue peptide (P51) homologous to human VAMP2 (residues 44-94) was examine d. Human VAMP2 is an 18-kDa protein located on the external membrane of sma ll synaptic vesicles and is targeted by four of the seven botulinum neuroto xin (BoNT) serotypes (B, D, F and G), The antiserum, designated anti-P51, r ecognized P51 but exhibited little cross-reactivity with the two cleavage p roducts that result from BoNT/B-mediated proteolysis of P51, The larger of these fragments, designated as P33 (residues 44-76), exhibited a weak but m easurable interaction with the antiserum. The smaller cleavage product, des ignated 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 P 51 using an indirect ELISA, The serine protease inhibitor phenylmethylsulfo nyl fluoride did not inhibit BoNT/B activity, but the zinc chelator N,N,N', N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) and the elastase inhibi tor 7-N-phenylcarbamoylamino-4-chloro-3-propyloxyisocoumarin (ICD 1578) pro duced complete blockade of BoNT/B LC action, Under ideal conditions, it wil l be possible to evaluate up to seven candidate anti-BoNT/B drugs in tripli cate at four concentrations using a single 96-well microtiter plate. These findings indicate that the ELISA will be suitable for rapid screening of Bo NT/B inhibitors.