ELISA Protocol - SEC

ELISA protocol - SEC

A newly designed brief (3.5 h) and simple ELISA protocol was used to quantify the extracellular concentration of SEC in TSB and milksamples. A microtiter plate (Costar 9018 polystyrene, Flat Bottom 96-Well Assay Microplate; Corning Inc., USA) was coated with 100 µl/well of a solution containing 90 ng/mL anti-SEC affinity-purified polyclonal antibody from sheep (SLCI111, Toxin Technology Inc., USA) in carbonate-bicarbonate buffer (0.05 M carbonate buffer, pH 9.6, Sigma-Aldrich) and the covered plate was incubated at 4 °C overnight. The next day, the coating solution was removed and the plate was washed 4 times with 200 µl/well of washing buffer (PBST; 0.05% Tween 20 in 10 mM PBS pH 7.4, Sigma-Aldrich). Standards, milk samples or culture supernatants were added into the plate (100 µl/well) at appropriate dilutions (non-diluted, 10x, 100x) and incubated for 90 min at 37 °C with constant shaking at 250 rpm (PST-60HL-4, Plate Shaker-Thermostat, BioSan SIA, Latvia). The standard curve was prepared by using serial dilutions of highly purified SEC (CT111, Toxin Technology Inc., USA) to specific concentrations. Serial dilution of samples was performed in the same medium as was used in given experiment. The range of the concentration was from 10 to 2500 pg/mL (diluted in milk and TSB). The plate was washed four times and the biotinylated antibody (SBCC111, Toxin Technology Inc., USA; diluted 2000× in PBST buffer) was added (100 µl/well). After incubation the plate was incubated for 30 min at 37 °C with shaking and washed. NeutrAvidin™ linked alkaline phosphatase (Pierce™ NeutrAvidin™, Alkaline Phosphatase Conjugated (1.7 mg/mL), Thermo Scientific, USA;diluted 1000× in PBST buffer) was added (100 µl/well) and the plate was incubated for 40 min at 37 °C with shaking. The plate was then washed and substrate (SIGMAFAST™ pNPP tablets, Sigma-Aldrich) added (100 µl/well). After incubation for 15 min in darkness (covered with foil) the color was developed to yellow in positive wells. The reaction was stopped with 50 µl per well of 4 M NaOH and absorbance was measured at 405 nm (Tecan Group Ltd., Switzerland). Accurate absorbance values from 0.0 to 2.0 OD (mean of three technical repetitions wells) were plotted against enterotoxin concentration and values of samples were determined using logarithmic regression. The detection limit of the ELISA was 10 pg/mL, only these average values lower than LOQ are not in the linear area for determining the concentration toxin.

ELISA protocol - SEA

A newly designed brief (3.5 h) and simple ELISA protocol was used to quantify the extracellular concentration of SEC in TSB and milksamples. A microtiter plate (Costar 9018 polystyrene, Flat Bottom 96-Well Assay Microplate; Corning Inc., USA) was coated with 100 µl/well of a solution containing 160ng/mL anti-SEC affinity-purified polyclonal antibody from sheep (SLAI101, Toxin Technology Inc., USA) in carbonate-bicarbonate buffer (0.05 M carbonate buffer, pH 9.6, Sigma-Aldrich) and the covered plate was incubated at 4 °C overnight. The next day, the coating solution was removed and the plate was washed 4 times with 200 µl/well of washing buffer (PBST; 0.05% Tween 20 in 10 mM PBS pH 7.4, Sigma-Aldrich). Standards,milk samples or culture supernatants were added onto the plate (100 µl/well) at appropriate dilutions (non-diluted, 10x, 100x) and incubated for 60 min at 37 °C with constant shaking at 250 rpm (PST-60HL-4, Plate Shaker-Thermostat, BioSan SIA, Latvia). The standard curve was prepared by using serial dilutions of highly purified SEC (AT101, Toxin Technology Inc., USA) to specific concentrations. Serial dilution of samples was performed in the same medium as was used in given experiment. The range of theconcentration was from 20 to 5000 pg/mL for TSB and 20 pg/mLto 20ng/mL for milk. After incubation the plate was washed four times and the biotinylated antibody (SBAC101, Toxin Technology Inc., USA; diluted 6000× in PBST buffer) was added (100 µl/well). The plate was incubated for 45 min at 37 °C with shaking and washed. NeutrAvidin™ linked alkaline phosphatase (Pierce™ NeutrAvidin™, Alkaline Phosphatase Conjugated (1.7 mg/mL), Thermo Scientific, USA;diluted 1000× in PBST buffer) was added (100 µl/well) and the plate was incubated for 45 min at 37 °C with shaking. The plate was then washed and substrate (SIGMAFAST™ pNPP tablets, Sigma-Aldrich) added (100 µl/well). After incubation for 20 min in darkness (covered with foil) the color was developed to yellow in positive wells. The reaction was stopped with 50 µl per well of 4 M NaOH and absorbance was measured at 405 nm (Tecan Group Ltd., Switzerland). Accurate absorbance values from 0.0 to 2.0 OD (three technical repetitions) were plotted against enterotoxin concentration and values of samples were determined using logarithmic regression. The detection limit of the ELISA was 15 pg/mL, only these average values lower than LOQ are not in the linear area for determining the concentration toxin.