CC49 Antibody Modification with Tetrazine-NHS

A CC49 solution (2 mg from a 5 mg/mL solution) reacted with methyl-tetrazine-NHS¹⁹ (30 equiv, 4.8 μL in dimethyl sulfoxide) in a total volume of 500 μL PBS. Sodium carbonate buffer (1 M) was used to adjust the pH to 9, and the reaction was carried out for 45 min at RT in the dark. The purification of the conjugate was performed via size exclusion chromatography (PD-10 column) and concentrated using vivaspin2 centrifuge tubes in order to be stored in PBS at 4 °C. To determine the tetrazine loading into the antibody, MS using a Q-TOF instrument was used providing an average of 8 tetrazine molecules per antibody (Figure S2), as previously described.¹⁹