High-Performance Near-Infrared Fluorescent Secondary Antibodies for Immunofluorescence.

A broad array of imaging and diagnostic technologies employs fluorophore-labeled antibodies for biomarker visualization, an experimental technique known as immunofluorescence. Significant performance advantages, such as higher signal-to-noise ratio, are gained if the appended fluorophore emits near-infrared (NIR) light with a wavelength >700 nm. However, the currently available NIR fluorophore antibody conjugates are known to exhibit significant limitations, including low chemical stability and photostability, weakened target specificity, and low fluorescence brightness. These fluorophore limitations are resolved by employing a NIR heptamethine cyanine dye named s775z whose chemical structure is very stable, charge-balanced, and sterically shielded. Using indirect immunofluorescence for imaging and visualization, a secondary IgG antibody labeled with s775z outperformed IgG analogues labeled with the commercially available NIR fluorophores, IRDye 800CW and DyLight800. Comparison experiments include three common techniques: immunocytochemistry, immunohistochemistry, and western blotting. Specifically, the secondary IgG labeled with s775z was 3-8 times brighter, 3-6 times more photostable, and still retained excellent target specificity when the degree of antibody labeling was high. The results demonstrate that antibodies labeled with s775z can emit total photon counts that are 1-2 orders of magnitude higher than those currently possible, and thus enable unsurpassed performance for NIR fluorescence imaging and diagnostics. They are especially well suited for analytical applications that require sensitive NIR fluorescence detection or use modern photon-intense methods that require high photostability.