A neuronal cell-based botulinum neurotoxin assay for highly sensitive and specific detection of neutralizing serum antibodies

Abstract

Clostridium botulinum neurotoxin (BoNT) serotypes A and B are widely used as pharmaceuticals to treat various neurological disorders and in cosmetic applications. The major adverse effect of these treatments has been resistance to treatment after multiple injections. 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 procedure is commercially available that reliably tests for the presence of such antibodies. This report presents a highly sensitive and specific neuronal cell-based assay that provides sensitive and specific detection of neutralizing antibodies to BoNT/A.

Figure 1

Differentiation of primary rat spinal cord cells over time. Cells were plated onto collagen coated cover slips and observed by light microscopy for up to 25 days after plating.

Figure 2

Western blots showing BoNT sensitivity of RSC cells. RSC cells were exposed to serial dilutions of BoNT/A (a), E (b), and B (c), and cell lysates were examined by Western blot for SNAP25 (for A and E) or VAMP (for B) cleavage.

Figure 3

Protection against SNAP25 cleavage in RSC cells by human sera. RSC cells were exposed to A: a mixture of 125 pg BoNT/A and 25 % human serum of 15 different patients, and B: a mixture of 12.5 pg BoNT/A and 25 % of human serum #3 and #8, and the cell lysates were assayed for SNAP25 cleavage by Western blot. The +C represents toxin only controls and the −C represents cells not treated with toxin or serum.

Figure 4

Sensitivity of RSC cells for antibody detection. RSC cells were exposed to a mixture of 125 pg BoNT/A and a (1) 1:200, (2) 1:400, (3) 1:800, (4) 1:1600, (5) 1:3200 dilution of human serum #4. A Western blot of SNAP25 cleavage (a) and a quantitative representation of densitometric analysis of three Western blots (b) are shown. The +C represents 125 pg BoNT/A without serum, and the −C contains no toxin and no serum.