Versatile Protein Conjugation Protocols

All conjugations were performed in PBS (pH 7.4) after the Fab or scFv fragments with TAFs were concentrated to 50 µM using a 10-kDa cutoff centrifugal filter device (Millipore). Depending on the kind of modifications we need, different reagents were added. For modification of hydrazide-CY5, antibody fragments, hydrazide-CY5 (Cat No. BDC-42, purchased from Confluore, 10 mM in DMSO, 50 eq) and aniline (0.1 eq) were added to conjugation buffer (6.5 mM KH₂PO₄, 95 mM K₂HPO₄, 125 mM NaCl, pH=8.4) and incubated at 37 °C for 3 days. For other modifications, DBCO-CY5 (Cat No. BCD-34, purchased from Confluore, 10 mM in DMSO, 4 eq), DBCO-CY7 (Cat No. BCD-22, purchased from Confluore, 10 mM in DMSO, 4 eq), DBCO-CY3 (Cat No. BCD-30, purchased from Confluore, 10 mM in DMSO, 4 eq), DBCO-(PEG)3-VC-PAB-MMAE (Cat No. HY-111012, purchased from MedChemExpress, 30 mM in DMSO, 4 eq), TCO-PEG20K (Cat No. BGNH-45, purchased from Confluore, 20 mM in ddH₂O, 4 eq) and TCO-PEG40K (Cat No. BGNH-46, purchased from Confluore, 10 mM in ddH₂O, 4 eq), TCO-PEG3-NOTA (Cat No. R-056, purchased from Ruixibio, 100 mM in DMSO, 4 eq) was added to the solution of protein in one pot. The solution was incubated for 4 h at 25 °C. Upon completion, unreacted compound was removed by using a 10-kDa cutoff centrifugal filter device (Millipore) or Superdex 75 Increase 10/300 GL column (Cytiva). The conjugates in PBS were stored at 4 °C for short-term use and −80 °C in aliquots for long-term use. The concentration was determined by measuring the absorbance at 280 nm and Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).