RESUMO
INTRODUCTION: Botulinum neurotoxin type A (BoNTA) is one of seven serotypes produced by Clostridium botulinum (types A thru G) and is the serotype most widely used to treat both cosmetic and medical conditions. Potency for botulinum toxin preparations is expressed in mouse LD50 units. There is a need to develop a non-animal based replacement for this potency assay. METHODS: An in vitro potency assay measuring BoNTA activity has been developed that addresses both BoNTA heavy chain binding to its cell receptor SV2C and BoNTA light chain enzymatic activity in cleaving SNAP-25, an intracellular protein essential in neurotransmitter release. This bifunctional assay utilizes a 96 well microtiter format and well defined reagents. Assay characterization determined that the relative standard deviation for intermediate precision was less than 10%. RESULTS: The assay standard curve covers the range of BoNTA concentrations from 0.0624 to 32 ng/mL. Specificity was demonstrated with purified BoNTA heavy chain which inhibited the activity in a dose dependent manner. A correlation between this bifunctional assay and the mouse LD50 potency assay was demonstrated.
Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Ensaios Enzimáticos/métodos , Animais , Toxinas Botulínicas Tipo A/antagonistas & inibidores , Toxinas Botulínicas Tipo A/isolamento & purificação , Toxinas Botulínicas Tipo A/toxicidade , Dose Letal Mediana , Camundongos , Especificidade por SubstratoRESUMO
Typically, the removal of pyroglutamate from the protein chains of immunoglobulins with the enzyme pyroglutamate aminopeptidase requires the use of chaotropic and reducing agents, quite often with limited success. This article describes a series of optimization experiments using elevated temperatures and detergents to denature and stabilize the heavy chains of immunoglobulins such that the pyroglutamate at the amino terminal was accessible to enzymatic removal using the thermostable protease isolated from Pyrococcus furiosus. The detergent polysorbate 20 (Tween 20) was used successfully to facilitate the removal of pyroglutamate residues. A one-step digestion was developed using elevated temperatures and polysorbate 20, rather than chaotropic and reducing agents, with sample cleanup and preparation for Edman sequencing performed using a commercial cartridge containing the PVDF membrane. All of the immunoglobulins digested with this method yielded heavy chain sequence, but the extent of deblocking was immunglobulin dependent (typically>50%).