High-Performance Near-Infrared Fluorescent Secondary Antibodies for Immunofluorescence

Abstract

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.

Comparison of cRGDfK Peptide Probes with Appended Shielded Heptamethine Cyanine Dye (s775z) for Near Infrared Fluorescence Imaging of Cancer

“…Recently, the NHS ester of s775z has become commercially available as a reagent for amidebased bioconjugation, 58 and it has been used to label lysine residues on the surface of antibodies to give NIR fluorescent antibodies with an unsurpassed combination of high photostability and fluorescence brightness. 22 This present contribution reports two new reactive homologues, namely, s775zmaleimide which reacts with a thiol group, and s775z-alkyne which reacts with an azide group. Three cancer-targeting NIR fluorescent probes (s775z-aRGD, s775z-tRGD, and s775z-sRGD) were prepared, each with an appended cRGDfK peptide to provide a selective affinity for integrin receptors that are overexpressed in many tumors.…”

“…57 ■ CONCLUSIONS This is the third research publication describing the favorable properties of sterically shielded NIR fluorescent cyanine dye s775z (Scheme 1). 21,22 The chemical structure of s775z has two short shielding arms that prevent dye self-aggregation, but they do not block association of bioconjugates with their targets. In addition, s775z possesses a geometric distribution of opposing charge that lowers association with off-target proteins and cell membranes, which in turn minimizes nonspecific cell permeation and cytotoxicity.…”

“…In addition, s775z possesses a geometric distribution of opposing charge that lowers association with off-target proteins and cell membranes, which in turn minimizes nonspecific cell permeation and cytotoxicity. Recently, the NHS ester of s775z has become commercially available as a reagent for amide-based bioconjugation, and it has been used to label lysine residues on the surface of antibodies to give NIR fluorescent antibodies with an unsurpassed combination of high photostability and fluorescence brightness . This present contribution reports two new reactive homologues, namely, s775z-maleimide which reacts with a thiol group, and s775z-alkyne which reacts with an azide group.…”

“…We have contributed to this Cy7 probe optimization effort by designing a new class of heptamethine cyanine dyes that we call shielded Cy 7 dyes. , The prototype example is s775z (Scheme ), which has two short triethylene glycol arms (colored red) that shield each face of the heptamethine fluorochrome (colored blue). The shielding arms prevent dye self-aggregation, but they do not weaken association of bioconjugates with their targets.…”

A Series of PSMA-Targeted Near-Infrared Fluorescent Imaging Agents

“…Our qualitative, head-to-head comparison of DyLight800-3 to YC-27 did not reveal a substantial difference in tumor uptake, although the latter had significantly lower renal uptake. The literature also provides little evidence for the superiority of DyLight800 over IRDye 800CW, and actually suggests the contrary in terms of quantum yield and brightness in certain in vitro assays [39,40]. Remaining, however, is the possibility that the issue of coupling a particular dye with a certain camera may further enhance sensitivity, while we used the Pearl Impulse Imager for our studies herein.…”