Goat anti rabbit igg conjugated with horseradish peroxidase

The ability of LTB protein to bind to gangliosides is indication of immunogenecity. 7, 8, 10 The microwell plate was coated with 100 μl/well of 3 μg/ml GM1 ganglioside (Sigma-Aldrich) in bicarbonate buffer, pH 9.6 at 4°C overnight. After three washes with PBST, the wells were blocked with 1% BSA in 0.01M PBS (300 µl/well) at 37°C for 2 h. The wells were washed three times with PBST and then incubated with the protein extract (100 μl/well) from the LTB transgenic carrot hairy roots for 2 h at 37°C. The wells were coated with 100 μl/well of 3.0 μg/ml BSA as a control. For the primary and secondary antibody treatments, the wells were incubated with a 1:5000 dilution of rabbit anti-LTB antibody (Sigma-Aldrich) (100 μl/well) in 0.01M PBS containing 0.5% BSA for 2 h at 37°C and washed four times with PBST. Subsequently, the wells were incubated with a 1:10000 dilution of goat anti-rabbit IgG conjugated with horseradish peroxidase (Sigma-Aldrich) (100 μl/ well) in 0.01M PBS containing 0.5% BSA for 2 h at 37°C and washed four times with PBST. Finally, the plate was incubated with100 μl/well TMB substrates (Sigma-Aldrich) for 30 min at RT in the dark. After incubation, the reaction was measured at an absorbance of 620 nm in an automated ELISA system (BioEra).