Cell-free measurements of brightness of fluorescently labeled antibodies.
Validation of imaging contrast agents, such as fluorescently labeled imaging antibodies, has been recognized as a critical challenge in clinical and preclinical studies. As the number of applications for imaging antibodies grows, these materials are increasingly being subjected to careful scrutiny. Antibody fluorescent brightness is one of the key parameters that is of critical importance. Direct measurements of the brightness with common spectroscopy methods are challenging, because the fluorescent properties of the imaging antibodies are highly sensitive to the methods of conjugation, degree of labeling, and contamination with free dyes. Traditional methods rely on cell-based assays that lack reproducibility and accuracy. In this manuscript, we present a novel and general approach for measuring the brightness using antibody-avid polystyrene beads and flow cytometry. As compared to a cell-based method, the described technique is rapid, quantitative, and highly reproducible. The proposed method requires less than ten microgram of sample and is applicable for optimizing synthetic conjugation procedures, testing commercial imaging antibodies, and performing high-throughput validation of conjugation procedures.
Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Haiying
H
Zhou
Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
George
G
Tourkakis
Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Dennis
D
Shi
Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
David M
DM
Kim
Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Hairong
H
Zhang
Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Tommy
T
Du
Department of Medicine and Siteman Cancer Center Flow Cytometry Core, Washington University School of Medicine, St. Louis, MO 63110, USA.
William C
WC
Eades
Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Mikhail Y
MY
Berezin
D002474:Cell-Free System; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D008163:Luminescent Measurements / Q000379:methods; D008856:Microscopy, Fluorescence; D013194:Staining and Labeling
D005456:Fluorescent Dyes; D007074:Immunoglobulin G