SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E

Abstract

Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay.

Keywords: BoNT/A; BoNT/E; SNAP-25; SiMa cells; capture ELISA; cell-based assay; immunodetection; toxin neutralization test (TNT).

Figure 1

Bright field relief contrast images of SiMa cells pre- and post-differentiation, and post-treatment with BoNT. SiMa cells were initially cultured in basal medium (a), and then differentiated in serum-free medium on Biocoat™ 6-well plates in the presence of 25 µg/mL GT1b for 72 h (b). Differentiated cells after treatment with either BoNT/A (c) or BoNT/E (d), both at 200 LD50/mL for a further 48 h. Scale bars represent 50 µm.

Figure 2

Dose dependent detection of cleaved SNAP-25 specific for (a) Botulinum toxins (BoNT)/A and (b) BoNT/E toxins. SiMa cells were differentiated for 3 days on 96-well tissue culture plates and treated with either purified BoNT/A or BoNT/E toxins in a range of concentrations between 1–1280 LD50/mL (~5 pg/mL to ~5 ng/mL). After 48 h exposure, cells were lysed and subjected to toxin specific capture ELISA for detection of either BoNT/A (a) or BoNT/E (b) cleaved SNAP-25. Dotted line indicates controls where cells were not exposed to toxins. Results are from one typical assay performed on at least three independent occasions and each data set is a mean from four individual wells ±SD. (c) Schematic overview of capture ELISA for BoNT/A and BoNT/E: BoNT/A cleaves SNAP-25 between amino acids 197 and 198 and the cleavage product is captured using a specific neo-epitope antibody raised against a peptide corresponding to amino acids 190–197 of SNAP-25 (SNAP-25_190–197). BoNT/E cleaves SNAP-25 between amino acids 180 and 181 and the cleavage product is captured using a specific neo-epitope antibody raised against a peptide corresponding to amino acids 173–180 of SNAP-25 (SNAP-25_173–180). The captured cleavage product is then detected using two polyclonal detection antibodies that bind to two distinct sites, SNAP-25_1–57 and SNAP-25_111–157.

Figure 2

Dose dependent detection of cleaved SNAP-25 specific for (a) Botulinum toxins (BoNT)/A and (b) BoNT/E toxins. SiMa cells were differentiated for 3 days on 96-well tissue culture plates and treated with either purified BoNT/A or BoNT/E toxins in a range of concentrations between 1–1280 LD50/mL (~5 pg/mL to ~5 ng/mL). After 48 h exposure, cells were lysed and subjected to toxin specific capture ELISA for detection of either BoNT/A (a) or BoNT/E (b) cleaved SNAP-25. Dotted line indicates controls where cells were not exposed to toxins. Results are from one typical assay performed on at least three independent occasions and each data set is a mean from four individual wells ±SD. (c) Schematic overview of capture ELISA for BoNT/A and BoNT/E: BoNT/A cleaves SNAP-25 between amino acids 197 and 198 and the cleavage product is captured using a specific neo-epitope antibody raised against a peptide corresponding to amino acids 190–197 of SNAP-25 (SNAP-25_190–197). BoNT/E cleaves SNAP-25 between amino acids 180 and 181 and the cleavage product is captured using a specific neo-epitope antibody raised against a peptide corresponding to amino acids 173–180 of SNAP-25 (SNAP-25_173–180). The captured cleavage product is then detected using two polyclonal detection antibodies that bind to two distinct sites, SNAP-25_1–57 and SNAP-25_111–157.

Figure 3

Dose dependent inhibition of BoNT/A cleavage of SNAP-25 from SiMa cells by (a) reference polyclonal and (b) humanized recombinant monoclonal antibodies against BoNT/A. SiMa cells were differentiated for 3 days on 96-well tissue culture plates and treated with a mixture of purified BoNT/A toxin (200 LD50/mL or 40LD50 per well) and (a) reference antitoxin for BoNT/A (NIBSC product code 59/021) in the range of concentrations between 0.1 IU and 0.1 mIU or (b) humanized monoclonal antibodies targeting heavy chain (HC) or the light chain (LC) of BoNT/A [12]. After 48 h exposure to the corresponding mixtures, cells were lysed and subjected to capture ELISA for detection of BoNT/A cleaved SNAP-25. Results are from one representative assay where each data set is the mean from two individually treated wells ±SD. Reference antitoxin for BoNT/A (NIBSC product code 59/021) was also included as a negative control in the absence of BoNT/A (a).

Figure 4

Specificity of SiMa cell toxin neutralization assay for BoNT/A. SiMa cells were differentiated for 3 days on 96-well tissue culture plates and treated with a mixture of purified BoNT/A toxin (200 LD50/mL or 40 LD50 per well) and (a) a reference antitoxin for BoNT/E (NIBSC product code 02/318) in the range between 1 IU and 0.1 mIU or (b) humanized recombinant monoclonal antibody targeting the LC of BoNT/E (ELC18) [13]. After 48 h exposure to the corresponding mixtures, cells were lysed and subjected to capture ELISA for detection of BoNT/A cleaved SNAP-25. Results are from a representative experiment where each data set is the mean from two individually treated wells ±SD. Reference antitoxin for BoNT/E (NIBSC product code 02/318) and humanized antibody ELC18 were also incubated with SiMa cells in the absence of BoNT/A as negative controls and were from a single cell reading per dilution.

Figure 5

Dose dependent inhibition of BoNT/E cleavage of SNAP-25 from SiMa cells by polyclonal type E antitoxins. SiMa cells were differentiated for 3 days on 96-well tissue culture plates and treated with a mixture of purified BoNT/E toxin (200 LD50/mL or 40 LD50 per well) and reference antitoxin for BoNT/E (NIBSC product code 02/318), or two separate batches of polyclonal trivalent antitoxin (#079012A and #081021A, with assumed potency of >50 IU/mL for antitoxin type E) diluted in the range between 0.1 IU/mL and 0.05 mIU/mL. Reference antitoxin for BoNT/A (NIBSC product code 59/021) was included as a negative control. After 48 h exposure to the corresponding mixtures, cells were lysed and subjected to capture ELISA for detection of BoNT/E cleaved SNAP-25. Results are from a single experiment where each data set is the mean of two individually treated wells ±SDs. SiMa cells were also treated with the antitoxins in the absence of BoNT/E and no signal was observed in the capture ELISA (Supplementary Materials Figure S1).