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Updated: Aug 23, 2026

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology
Published on: March 12, 2015
Development of a spectroscopic assay for bifunctional ligand-protein conjugates based on copper
Erik D Brady1, Hyun-Soon Chong, Diane E Milenic
1Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA. bradye@mail.nih.gov
Abstract:
A simple, non-radioactive method for the determination of ligand-to-protein ratio (L/P) for novel ligand-antibody conjugates has been developed based on an exchange equilibrium with the purple Cu(II) complex of arsenazo III. The method requires a UV/Vis spectrometer and has been verified for monoclonal antibody Herceptin conjugates of a variety of ligand modalities, including common macrocyclic compounds NOTA and TETA, and with a new bifunctional tachpyridine (1H-Pyrrole-1-butanamide,N-[4-[[(1alpha,3alpha,5alpha)-3,5-bis[(2-pyridinylmethyl)amino]cyclohexyl](2-pyridinylmethyl)amino]butyl]-2,5-dihydro-2,5-dioxo-(9CI)). The spectroscopically derived values for L/P were verified by titration of the ligand-antibody conjugate with 64Cu. In each case, the value obtained by UV/Vis spectroscopy matches that found by radiolabeling. The method is rapid, taking less than 30 minutes with each ligand in this study.
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