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 mo

SUBMITTER: Szabo A 

PROVIDER: S-EPMC5985035 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature