Optimal labeling condition of antibodies available for immunofluorescence endoscopy

In recent years, labeled antibodies have been used for diagnostic imaging in many studies. In this study, we investigated the mode of binding in antibodies labeled with ICG derivatives newly developed for the diagnosis of microcarcinomas, and evaluated the optimal binding molar ratio between the labeling compounds and antibody.

MUC 1 antibody and ICG derivatives (ICG-ATT and ICG-sulfo-OSu) were used. ICG derivatives non-covalently bound to the antibody were removed with ethyl acetate, and the ratio of ICG derivatives covalently bound to the labeled antibody was confirmed. During purification of the labeled antibody, the amount of each labeling compound reacting with 1 molecule of the antibody varied as follows: 4, 8, 16, and 32 molar equivalents. Subsequently, the intensity of fluorescence was evaluated by spectroscopy and infrared fluoroscopy.

The ratio of residual ICG derivative labeling the antibody was 67.4% for ICG-ATT and 65.0% for ICG-sulfo-OSu. When fluorescent antibody labeled with ICG-ATT at an F/P ratio of 2.94 or 4.18 was used, specific and clear fluorescent images of the antigen were obtained. When ICG-ATT-labeled antibody at an F/P ratio of 6.50 or 6.75 was used, the fluorescence intensity decreased and the fluorescent images of antigen became unclear.

It was found that the ICG-ATT-labeled antibody was a more specific and sensitive marker than ICG-sulfo-OSu-labeled antibody, and that lower binding molar ratios of ICG-ATT were more useful for labeling the antibody.