Rapid microplate assay for monitoring botulinum neurotoxin B catalytic activity

✍ Scribed by James E. Keller; Janet L. Nowakowski; Margaret G. Filbert; Michael Adler

• Book ID: 101288673
• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 67 KB
• Volume: 19
• Category: Article
• ISSN: 0260-437X
• DOI: 10.1002/(sici)1099-1263(199912)19:1+<s13::aid-jat607>3.0.co;2-3

✦ Synopsis

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/Bmediated 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.