The Effect of Fluorophore Conjugation on Antibody Affinity and the Photophysical Properties of Dyes

Abstract

Because the degree of labeling (DOL) of cell-bound antibodies, often required in quantitative fluorescence measurements, is largely unknown, we investigated the effect of labeling with two different fluorophores (AlexaFluor546, AlexaFluor647) in a systematic way using antibody stock solutions with different DOLs. Here, we show that the mean DOL of the cell-bound antibody fraction is lower than that of the stock using single molecule fluorescence measurements. The effect is so pronounced that the mean DOL levels off at approximately two fluorophores/IgG for some antibodies. We developed a method for comparing the average DOL of antibody stocks to that of the isolated, cell-bound fraction based on fluorescence anisotropy measurements confirming the aforementioned conclusions. We created a model in which individual antibody species with different DOLs, present in an antibody stock solution, were assumed to have distinct affinities and quantum yields. The model calculations confirmed that a calibration curve constructed from the anisotropy of antibody stocks can be used for determining the DOL of the bound fraction. The fluorescence intensity of the cell-bound antibody fractions and of the antibody stocks exhibited distinctly different dependence on the DOL. The behavior of the two dyes was systematically different in this respect. Fitting of the model to these data revealed that labeling with each dye affects quantum yield and antibody affinity differentially. These measurements also implied that fluorophores in multiply labeled antibodies exhibit self-quenching and lead to decreased antibody affinity, conclusions directly confirmed by steady-state intensity measurements and competitive binding assays. Although the fluorescence lifetime of antibodies labeled with multiple fluorophores decreased, the magnitude of this change was not sufficient to account for self-quenching indicating that both dynamic and static quenching processes occur involving H-aggregate formation. Our results reveal multiple effects of fluorophore conjugation, which must not be overlooked in quantitative cell biological measurements.

Multiple-labeled antibodies behave like single emitters in photoswitching buffer

“…Particularly noteworthy here is the observed strong unspecific background labeling of higher DOL antibodies, which has also been seen in our FLIM experiments ( Fig. 2b) corroborating again the finding that multiple-labeled antibodies exhibit a decreased binding affinity 20 . To investigate the blinking performance of multiple Al647-labeled antibodies in more detail we determined the average on-and off-times per blinking event as well as the fluorescence intensities (photons ms -1 ) in the on-state under different experimental conditions (Supplementary Table 2).…”

“…2d). A closer look at the FLIM images reveals that some of the DOL 8.3 antibodies label also other intracellular structures, which corroborates previous studies showing that multiple labeled antibodies exhibit a decreased affinity 20 . Interestingly, these unspecific binding antibodies exhibit a longer fluorescence lifetime, which can seriously complicate the interpretation of FLIM imaging experiments.…”

“…The DOL of commercially available antibodies has been optimized to 2-5 to minimize quenching effects and retain a high brightness and specific binding of the fluorescent probe 16,19,20 . Normally Table 1, Supplementary Table 2, Figure 3 and Supplementary Fig.…”

“…Since a typical IgG antibody carries numerous lysine residues it can be labeled with multiple dyes 18 and represents thus a multiple dye-labeled probe. However, the degree of labeling (DOL) has to be kept below 6-8 dyes per antibody to minimize quenching effects and retain a high binding specificity and affinity 19,20 . Usually antibodies are optimized for high brightness and efficient binding to their corresponding epitopes, which typically results in degrees of labeling (DOL) of 2-5 16,20 .…”

Fluorescently Labelled Silica Coated Gold Nanoparticles as Fiducial Markers for Correlative Light and Electron Microscopy

“… 15 (fluorescein in Stöber silica) and for fluorophores on antibodies by Szabó et al . 18 . An explanation for these observations can be found by taking into account concentration effects including self quenching.…”