Development of a Plasma Spot Assay for Detection of Fluorophore-labeled Antibodies in Mouse Imaging Studies

In vivo and ex vivo imaging of fluorescently labeled therapeutic antibodies (Ab-F) and antibody-drug conjugates (ADC-F) in mice provides an efficient approach for evaluating target-specific and nonspecific retention in tissues, inflammatory sites, and tumors. However, comparisons across compounds can be confounded by differences in pharmacokinetics, fluorophore brightness, and degree of labeling, all of which influence fluorescence intensity in regions of interest.

We developed a fluorescent plasma spot assay (FPSA) to measure relative Ab-F fluorescence in plasma and enable standardization of tissue fluorescence obtained during optical imaging studies.

FPSA measurements were consistent with Ab-F concentration and degree-of-labeling data generated by conventional analytical methods, including immunoassay and spectrophotometry. Additionally, the FPSA was used to determine fluorophore type and DOL conditions with minimal effects on Ab-F clearance and tissue retention to minimize in-study artifacts.

The FPSA can be performed concurrently with in vivo or ex vivo imaging, requires only small plasma volumes without additional processing, and allows plasma and tissue samples to be analyzed on the same instrument using matched fluorescence units. Integration of the FPSA with optimized labeling conditions provides an accurate, efficient method for comparing tissue retention of Ab-F and ADC-F in mouse optical imaging biodistribution studies.