Cell-free measurements of brightness of fluorescently labeled antibodies

Abstract

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.

Efficiency scale for scattering luminescent particles linked to fundamental and measurable spectroscopic properties

“…4 and 6 ), dye-dye interactions, and dye-specific properties like the oscillator strength and in the respective environment. Earlier, we calculated the particle brightness from the product of the number of molecules per particle, the corresponding ε of the dye determined in a model solvent such as BOB, and the measured of the particle dispersion ( ) 27 , 71 , see Fig. 7 (left panel).…”

Enhanced optical imaging and fluorescent labeling for visualizing drug molecules within living organisms

“…Many additional fluorescently labeled small molecule drugs, antibodies 110 , 111 , peptides 112 , nucleic acids 113 and other molecules have been developed for the illumination and treatment of lesions 114 . However, the current visualization strategies based on fluorophore–molecule conjugates may lower drug efficacy, increase off-target effects, influence molecular interactions, and lead to other unpredictable effects, such as false positive results due to premature release of the fluorophore from the compound.…”

Absolute Quantification of Protein Copy Number in Single Cells With Immunofluorescence Microscopy Calibrated Using Single-Molecule Microarrays

“…For absolute quantification, immunofluorescence is a much greater challenge owing to the problems in performance, particularly specificity, and variability of any antibody (performance, structure, and affinity), access to the target protein epitope that may be fully or partially blocked, the presence of any post-translational modifications on the target protein, and the cross-reactivity of the antibody for the nontarget species. The issues that antibodies can present in general have been captured in a number of thought-provoking reports. − Furthermore, the choice of the fluorophore and their number, and by how much this varies, conjugated to the antibody may also need to be taken into consideration. , It quickly becomes apparent, then, that absolute quantification is much harder to achieve in practice.…”