In vivo immunogenicity assay

Balb/c mice were used to evaluate the ADA titers of the different antibodies. Five mice per group were first immunized with 70 μg antibodies in Complete Freund’s Adjuvant and boosted with 70 μg antibodies in Incomplete Freund’s Adjuvant four times in two weeks. Four weeks after the first immunization, tail bleeds from the antibody-immunized mice were tested for ADA titers by indirect Enzyme-linked Immunoassay (ELISA). Briefly, each well of the 96-well high binding EIA plates was coated with 1 µg/mL of Atezolizumab or Maxatezo at 4 °C overnight in PBS. The serum samples were first incubated with 1% human sera for one hour at room temperature to block nonspecific mouse anti-human IgG antibodies. After two washes with PBS and blocking with 5% skim milk in PBS for 1 h at room temperature, wells were incubated with pretreated serum in 5% skim milk in PBS for another hour at room temperature. After two washes with PBS, wells were then incubated with HRP-conjugated goat anti-mouse IgG Fc-specific secondary antibodies (Jackson Lab) in 5% skim milk in PBS for 1 h at room temperature. After five washes with PBS containing 0.1% Tween 20 (PBST), HRP substrate 3, 3´, 5, 5´-tetramethylbenzidine (TMB) solution was added. The reaction was stopped with 0.1 M H₂SO₄ after 30 min and absorbance was measured at 450 nm with a microplate reader.