ML Streptomycin. Neuro-2a- Murine neuroblastoma (CCL-131; ATCC) were cultured in Costar flasks in ATCC’s recommended medium. Differentiation medium: EMEM with 2 mM GlutaMAXTM, 0.1 mM NEAA, 10 mM HEPES, 16 N2 supplement, and 16 B27 supplement. SH-SY5Y- Human neuroblastoma (94030304; ECACC) were cultured in Costar tissue culture flasks in ECACC’s recommended medium. Differentiation medium: EMEM with 2 mM GlutaMAXTM, 0.1 mM NEAA, 10 mM HEPES, 16 N2 supplement, and 16 B27 supplement. N18- Mouse neuroblastoma6Rat glioma hybrid (88112301; ECACC) were cultured in Costar tissue culture flasks, 162 cm2 (CLS3150; Corning). Growth medium: DMEM with 2 mM glucose, 2 mM glutamine, and 10 heatinactivated FBS. LA1-55n- human neuroblastoma (06041203; ECACC) were cultured in Costar flasks in ECACC’s recommended medium. SiMa- human neuroblastoma (ACC 164, DSMZ) were cultured in collagen IV flasks in DSMZ’s recommended medium. Differentiation medium: Minimum Essential Medium with 2 mM GlutaMAXTM I with Earle’s salts, 0.1 mM NonEssential Amino-Acids, 10 mM HEPES, 16 N2 supplement, and 16 B27 supplement. For optimal differentiation, PC-12, Neuro2a, LA1-55n, and SiMa cells were plated in 96-well plates at 56104 cells/well in 100 mL differentiation medium for three days.Monoclonal antibody to SNAPMurine monoclonal Title Loaded From File Antibodies specific for SNAP25197 were generated with the (C)DSNKTRIDEANQ peptide utilizing standard immunization protocols. Antibodies to SNAP25197 were screened in WB and ELISA. Antibodies were affinity purified from ascites before use in the SPR and ELISA assays.Figure 7. Sandwich ELISA assay with chemiluminescence is as 1480666 sensitive as the ECL assay. A. Example of a representative experiment in which differentiated SiMa cells were Title Loaded From File treated with BoNT/A at concentrations from 0.03 to 25 pM for 24 h followed by 48 h incubation, and the lysates were evaluated in the optimized chemiluminescence read-out. The results obtained were similar to the ECL read-out (EC50,0.5 1676428 to 2 pM), excellent signal to background, and reproducibility of the replicates. B. Summary of optimized parameters for the chemiluminescence CBPA. Seven parameters comprising cell culture and ELISA read-out were specifically optimized for this assay by testing several conditions for each parameter. doi:10.1371/journal.pone.0049516.gSurface Plasmon Resonance Binding AnalysisExperiments were performed on a BIAcore 3000 instrument (GE Healthcare). Ligands, SNAP25134?97 and SNAP25134?06 peptides, were immobilized on a CM5 chip using amine coupling. Analytes, Anti-SNAP25197 2E2A6 (1.1 mg/mL stock solution, AGN) or MC-6053 (15 mg/mL stock solution, Protein A purified, Research and Diagnostics Antibodies) antibodies, were injected atSensitive Cell-Based Potency Assay for BoNT/AFigure 8. The CBPA can measure BoNT/A biological activity in BOTOXH vials. A. A custom medium to reconstitute BOTOXH vials (the nominal value of 100 U was used) was designed to overcome the hypertonicity caused by NaCl present in the formulation. The matrix for the subsequent dilutions was kept constant. Performance of the assay was improved resulting in better sensitivity (EC50 = 0.9 U/well) and higher efficacy of uptake. B. Two different lots of BOTOXH (the nominal value of 100 U was used) were evaluated in the CBPA. Data was analyzed in PLA 2.0 resulting in 0.82 relative potency (CI: 0.7?.1) indicating similar potency of both lots. C. A single lot of BOTOXH was tested by two operators (n = 8 and n = 9 independent experime.ML Streptomycin. Neuro-2a- Murine neuroblastoma (CCL-131; ATCC) were cultured in Costar flasks in ATCC’s recommended medium. Differentiation medium: EMEM with 2 mM GlutaMAXTM, 0.1 mM NEAA, 10 mM HEPES, 16 N2 supplement, and 16 B27 supplement. SH-SY5Y- Human neuroblastoma (94030304; ECACC) were cultured in Costar tissue culture flasks in ECACC’s recommended medium. Differentiation medium: EMEM with 2 mM GlutaMAXTM, 0.1 mM NEAA, 10 mM HEPES, 16 N2 supplement, and 16 B27 supplement. N18- Mouse neuroblastoma6Rat glioma hybrid (88112301; ECACC) were cultured in Costar tissue culture flasks, 162 cm2 (CLS3150; Corning). Growth medium: DMEM with 2 mM glucose, 2 mM glutamine, and 10 heatinactivated FBS. LA1-55n- human neuroblastoma (06041203; ECACC) were cultured in Costar flasks in ECACC’s recommended medium. SiMa- human neuroblastoma (ACC 164, DSMZ) were cultured in collagen IV flasks in DSMZ’s recommended medium. Differentiation medium: Minimum Essential Medium with 2 mM GlutaMAXTM I with Earle’s salts, 0.1 mM NonEssential Amino-Acids, 10 mM HEPES, 16 N2 supplement, and 16 B27 supplement. For optimal differentiation, PC-12, Neuro2a, LA1-55n, and SiMa cells were plated in 96-well plates at 56104 cells/well in 100 mL differentiation medium for three days.Monoclonal antibody to SNAPMurine monoclonal antibodies specific for SNAP25197 were generated with the (C)DSNKTRIDEANQ peptide utilizing standard immunization protocols. Antibodies to SNAP25197 were screened in WB and ELISA. Antibodies were affinity purified from ascites before use in the SPR and ELISA assays.Figure 7. Sandwich ELISA assay with chemiluminescence is as 1480666 sensitive as the ECL assay. A. Example of a representative experiment in which differentiated SiMa cells were treated with BoNT/A at concentrations from 0.03 to 25 pM for 24 h followed by 48 h incubation, and the lysates were evaluated in the optimized chemiluminescence read-out. The results obtained were similar to the ECL read-out (EC50,0.5 1676428 to 2 pM), excellent signal to background, and reproducibility of the replicates. B. Summary of optimized parameters for the chemiluminescence CBPA. Seven parameters comprising cell culture and ELISA read-out were specifically optimized for this assay by testing several conditions for each parameter. doi:10.1371/journal.pone.0049516.gSurface Plasmon Resonance Binding AnalysisExperiments were performed on a BIAcore 3000 instrument (GE Healthcare). Ligands, SNAP25134?97 and SNAP25134?06 peptides, were immobilized on a CM5 chip using amine coupling. Analytes, Anti-SNAP25197 2E2A6 (1.1 mg/mL stock solution, AGN) or MC-6053 (15 mg/mL stock solution, Protein A purified, Research and Diagnostics Antibodies) antibodies, were injected atSensitive Cell-Based Potency Assay for BoNT/AFigure 8. The CBPA can measure BoNT/A biological activity in BOTOXH vials. A. A custom medium to reconstitute BOTOXH vials (the nominal value of 100 U was used) was designed to overcome the hypertonicity caused by NaCl present in the formulation. The matrix for the subsequent dilutions was kept constant. Performance of the assay was improved resulting in better sensitivity (EC50 = 0.9 U/well) and higher efficacy of uptake. B. Two different lots of BOTOXH (the nominal value of 100 U was used) were evaluated in the CBPA. Data was analyzed in PLA 2.0 resulting in 0.82 relative potency (CI: 0.7?.1) indicating similar potency of both lots. C. A single lot of BOTOXH was tested by two operators (n = 8 and n = 9 independent experime.
