A novel method to quantify IRDye800CW fluorescent antibody probes ex vivo in tissue distribution studies

Background: We describe a new method for biodistribution studies with IRDye800CW fluorescent antibody probes. This method allows the quantification of the IRDye800CW fluorescent tracer in percentage of injected dose per gram of tissue (% ID/g), and it is herein compared to the generally used reference method that makes use of radioactivity. Methods: Cetuximab was conjugated to both the near-infrared fluorophore IRDye800CW and/or the positron emitter 89-zirconium, which was injected in nude mice bearing A431 human tumor xenografts. Positron emission tomography (PET) and optical imaging were performed 24 h post-injection (p.i.). For the biodistribution study, organs and tumors were collected 24 h p.i., and each of these was halved. One half was used for the determination of probe uptake by radioactivity measurement. The other half was homogenized, and the content of the fluorescent probe was determined by extrapolation from a calibration curve made with the injected probe. Results: Tumors were clearly visualized with both modalities, and the calculated tumor-to-normal tissue ratios were very similar for optical and PET imaging: 3.31 +/- 1.09 and 3.15 +/- 0.99, respectively. Although some variations were observed in ex vivo analyses, tumor uptake was within the same range for IRDye800CW and gamma ray quantification: 15.07 +/- 3.66% ID/g and 13.92 +/- 2.59% ID/g, respectively. Conclusions: The novel method for quantification of the optical tracer IRDye800CW gives similar results as the reference method of gamma ray quantification. This new method is considered very useful in the context of the preclinical development of IRDye800CW fluorescent probes for optical molecular imaging, likely contributing to the selection of lead compounds that are the most promising for clinical translation.