Synthesis of IR700-conjugated hYP218

Conjugation of dyes with monoclonal antibody was performed according to previous report [15]. In brief, hYP218 (1.0 mg, 13.3 nmol) was incubated with IR700 NHS ester (130.3 μg, 66.7 nmol) in 0.1 M Na₂HPO₄ (pH 8.6) at room temperature for 1 h. The mixture was purified with a Sephadex G25 column (PD-10; GE Healthcare, Piscataway, NJ, USA). The protein concentration was determined with Coomassie Plus protein assay kit (Thermo Fisher Scientific Inc, Rockford, IL, USA) by measuring the absorption at 595 nm with UV-Vis (8453 Value System; Agilent Technologies, Santa Clara, CA, USA). The concentration of IR700 was measured by absorption at 689 nm to confirm the number of fluorophore molecules per mAb. The synthesis was controlled so that an average of two IR700 molecules was bound to a single antibody. As a quality control for the conjugate, we performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The Conjugate was separated by SDS-PAGE with a 4–20% gradient polyacrylamide gel (Life technologies, Gaithersburg, MD). A standard marker (Crystalgen Inc., Commack, NY) was used as a protein molecular weight marker. After electrophoresis at 80 V for 2.5 h, the gel was imaged with a Pearl Imager (LI-COR Biosciences, Lincoln, Nebraska, USA) using a 700 nm fluorescence channel. We used diluted hYP218 as non-conjugated control. The gel was stained with Colloidal Blue staining to determine the molecular weight of conjugate.