A neuronal cell-based assay for highly sensitive detection of pharmaceutical preparations of BONT/A and patient neutralizing serum antibodies

Abstract

Clostridium botulinum neurotoxins (BoNTs) A and B are widely used as pharmaceuticals to treat various neurologic disorders and in cosmetic applications. The mouse lethality method is widely accepted for assay of therapeutic preparations of BoNTs, but it has well-known drawbacks including wide variability, the need for a large number of animals, and 2–4 days time requirements. We present a primary neuronal spinal cord assay that is capable of detection of BoNT with a sensitivity of less than 0.1 U. The neuronal assay is shown to be highly sensitive and specific for detection and quantitation of BoNT in pharmaceutical toxin preparations (Mentor Biologics). The assay measures the ratio of cleaved compared to full-length SNAP-25 by Western blot and densitometry analyses. Another development of the assay is the detection of neutralizing serum antibodies, since an adverse effect of clinical treatments of patients with BoNT has been resistance to treatment after multiple injections. In a double-blinded analysis, sera were evaluated from approximately 40 patients, and SNAP-25 cleavage was inhibited in the cohort of non-responders but not in the controls. Currently, patients receiving BoNT therapies and patients enrolled in clinical trials for new applications and/or new formulations of BoNTs are not routinely monitored for the formation of neutralizing antibodies, since no assay other than the mouse protection assay is scientifically accepted that reliably tests for the presence of such antibodies. We present results that highlight the utility of the neuronal assay for highly sensitive and specific detection of neutralizing antibodies to BoNT/A in patient sera. New assay platforms for detection of BoNT and antibodies in neuronal cells are also being investigated and are described.