ICG is one of the dyes that is used for determining cardiac output, hepatic function, and liver blood flow, as well as for ophthalmic angiography. It has a long excitation wavelength and emission wavelength of about 780 nm and 800 nm, respectively. Because of its long wavelength near the infrared region and low cytotoxicity, ICG is used to label antibodies for in vivo assay. However, fluorescent intensity after conjugation with protein is quite low because of the formation of H-dimer or an energy transfer to antibody molecules after excitation. Dr. Kobayashi and others reported that the use of SDS and betamecraptoethanol with the conjugate increases fluorescent intensity dramatically by diminishing hydrophobic p-p interactions and separation of IgG chains. They applied a treated ICG-conjugated daclizumab (humanized monoclonal antibody) and humanized anti-HER IgG2 monoclonal antibody for in vivo assay to specifically visualized tumors.
2. Add 6.8-68 nmol of ICG-Sulfo-OSu/DMSO solution to the antibody solution and incubate at room temperature for 30 minutes.
3. Purify the reaction mixture with a sephadex G50 column.