Re in place.Identification of BoNT/A sensitive SiMa cellsNeuronal-derived cell lines were obtained from the American Tissue Culture Collection (ATCC, 24 cell lines), European Collection of Cell Cultures (ECACC, 11 cell lines), and German Collection of Microorganisms and Cell Cultures (DSMZ, 7 cell lines) and screened for their sensitivity to BoNT/A. Differentiated Neuro-2a cells were previously identified as BoNT/A sensitive and served as comparison for screening additional cell lines. The primary screen was performed using BoNT/A complex at 0 and 1 nM with 6 h treatment followed by overnight incubation in toxin free medium to allow for cleavage of SNAP25. Samples were analyzed in Western blots (WB) using anti-SNAP25 antibodies (mAb SMI-81 or pAb S9684) that detect both SNAP25206 and SNAP25197 bands, allowing the calculation of cleaved SNAP25. The best cell lines for BoNT/A uptake were Neuro-2a, LA-1-55n, PC12, N18, and SiMa (Figure 2A and Figure 3A). Undifferentiated SiMa cells [48] were more sensitive to BoNT/A than undifferentiated Neuro-2a cells (Figure 2B) with 22.4 SNAP25 cleavage at 0.11 nM and 38 at 0.33 nM BoNT/A after overnight treatment, while no cleavage was detected on undifferentiated Neuro-2a cells under these treatment conditions. BoNT/A uptake by SiMa cells was 223488-57-1 compared to the other candidate cell lines using a SNAP25197 WB-assay (Figure 3A). Neuro-2a, PC12, LA-1-55n, and SiMa cells were differentiated inSensitive Cell-Based Potency Assay for BoNT/AFigure 1. Characterization of anti-SNAP25197 monoclonal antibody 2E2A6. Specificity demonstrated by lack of cross-reactivity towards SNAP25206. A. Neuro-2a cells were treated with BoNT/A (150 kDa) from 0.01 to 10 nM (duplicate wells) for 24 h. Western blot was performed with 2E2A6 antibody (1 mg/mL). No cross-reactivity with SNAP25206 (no bands observed in the 0 nM BoNT/A lanes) and no other non-specific bands were detected in the whole blot. B C. NT 157 Surface plasmon resonance (SPR) was 1676428 used to characterize the 2E2A6 antibody and to compare the binding affinity and binding kinetics of 2E2A6 and MC-6053. 2E2A6 bound SNAP25197 with high affinity and did not bind SNAP25206 at any of the concentrations tested up to 10 mM. MC-6053 was able to bind SNAP25206 with a KD of 240 nM. D. The Kd for 2E2A6 is tenfold lower than the Kd for MC-6053 as shown in the normalized graph resulting in better affinity. doi:10.1371/journal.pone.0049516.gSensitivity and specificity of SiMa cellsThe hallmarks of high affinity BoNT/A uptake are rapid binding and internalization combined with sensitivity to low toxin concentrations. Those are exemplified in embryonic spinal cord neurons (eSC) exposed to 0.4 pM BoNT/A for two days rendering 50 SNAP25 cleavage [39] and eSC treated with 500 pM BoNT/A for 4 min followed by 2.5 h incubation producing 10?20 SNAP25 cleavage [40]. SiMa cells detect BoNT/A activity at sub-pM toxin concentrations with 24 h treatments (Figure 3). To demonstrate fast uptake, differentiated SiMa cells were treatedwith 1 nM BoNT/A from 1 to 60 minutes. SiMa cells produced significant cleavage of SNAP25 over background after treatments as short as one minute (Figure 4A). The specificity of a method defines its ability to measure the analyte of interest and differentiate it from similar compounds. This CBPA is 1662274 specific to BoNT/A by design since the 2E2A6 monoclonal antibody only recognizes SNAP25197. To replace the bioassay, the CBPA must distinguish a fully active neurotoxin from al.Re in place.Identification of BoNT/A sensitive SiMa cellsNeuronal-derived cell lines were obtained from the American Tissue Culture Collection (ATCC, 24 cell lines), European Collection of Cell Cultures (ECACC, 11 cell lines), and German Collection of Microorganisms and Cell Cultures (DSMZ, 7 cell lines) and screened for their sensitivity to BoNT/A. Differentiated Neuro-2a cells were previously identified as BoNT/A sensitive and served as comparison for screening additional cell lines. The primary screen was performed using BoNT/A complex at 0 and 1 nM with 6 h treatment followed by overnight incubation in toxin free medium to allow for cleavage of SNAP25. Samples were analyzed in Western blots (WB) using anti-SNAP25 antibodies (mAb SMI-81 or pAb S9684) that detect both SNAP25206 and SNAP25197 bands, allowing the calculation of cleaved SNAP25. The best cell lines for BoNT/A uptake were Neuro-2a, LA-1-55n, PC12, N18, and SiMa (Figure 2A and Figure 3A). Undifferentiated SiMa cells [48] were more sensitive to BoNT/A than undifferentiated Neuro-2a cells (Figure 2B) with 22.4 SNAP25 cleavage at 0.11 nM and 38 at 0.33 nM BoNT/A after overnight treatment, while no cleavage was detected on undifferentiated Neuro-2a cells under these treatment conditions. BoNT/A uptake by SiMa cells was compared to the other candidate cell lines using a SNAP25197 WB-assay (Figure 3A). Neuro-2a, PC12, LA-1-55n, and SiMa cells were differentiated inSensitive Cell-Based Potency Assay for BoNT/AFigure 1. Characterization of anti-SNAP25197 monoclonal antibody 2E2A6. Specificity demonstrated by lack of cross-reactivity towards SNAP25206. A. Neuro-2a cells were treated with BoNT/A (150 kDa) from 0.01 to 10 nM (duplicate wells) for 24 h. Western blot was performed with 2E2A6 antibody (1 mg/mL). No cross-reactivity with SNAP25206 (no bands observed in the 0 nM BoNT/A lanes) and no other non-specific bands were detected in the whole blot. B C. Surface plasmon resonance (SPR) was 1676428 used to characterize the 2E2A6 antibody and to compare the binding affinity and binding kinetics of 2E2A6 and MC-6053. 2E2A6 bound SNAP25197 with high affinity and did not bind SNAP25206 at any of the concentrations tested up to 10 mM. MC-6053 was able to bind SNAP25206 with a KD of 240 nM. D. The Kd for 2E2A6 is tenfold lower than the Kd for MC-6053 as shown in the normalized graph resulting in better affinity. doi:10.1371/journal.pone.0049516.gSensitivity and specificity of SiMa cellsThe hallmarks of high affinity BoNT/A uptake are rapid binding and internalization combined with sensitivity to low toxin concentrations. Those are exemplified in embryonic spinal cord neurons (eSC) exposed to 0.4 pM BoNT/A for two days rendering 50 SNAP25 cleavage [39] and eSC treated with 500 pM BoNT/A for 4 min followed by 2.5 h incubation producing 10?20 SNAP25 cleavage [40]. SiMa cells detect BoNT/A activity at sub-pM toxin concentrations with 24 h treatments (Figure 3). To demonstrate fast uptake, differentiated SiMa cells were treatedwith 1 nM BoNT/A from 1 to 60 minutes. SiMa cells produced significant cleavage of SNAP25 over background after treatments as short as one minute (Figure 4A). The specificity of a method defines its ability to measure the analyte of interest and differentiate it from similar compounds. This CBPA is 1662274 specific to BoNT/A by design since the 2E2A6 monoclonal antibody only recognizes SNAP25197. To replace the bioassay, the CBPA must distinguish a fully active neurotoxin from al.
