Quantitative Assessment of Antibody Internalization with Novel Monoclonal Antibodies against Alexa Fluorophores

Abstract

Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface–bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor–labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

Detection of Subtle Immune Defects in Individuals at Risk of Pneumococcal Disease

“…Because αδdex on its own did not elicit up-regulation of CD25 (fig 3.2 A) or HLA-DR (fig 3.2 B) it can beconcluded that αδdex itself has no effect on CD4+ T-cell activation, at least not with respect to these particular activation markers. This conclusion was also drawn previously in our group (Preciado-Llanes et al, 2015).Receptor-mediated endocytosis occurs to an extent when using antibody labelling protocols(Liao-Chan et al, 2015, Ingle et al, 2008, especially when cell populations are stimulated and activated. Therefore it cannot be concluded whether stimulations actually reduced the frequency of CD19+ Bcells relative to the un-stimulated control (p ≤ 0.050; fig 3.3 A), or if receptor internalisation had occurred.…”

Quantitative characterization of the regulation of iron metabolism in glioblastoma stem‐like cells using magnetophoresis

“…The presence of surface epitopes can also be inconsistent due to cell cycle‐dependent expression as well as receptor recycling (Jaksch, Munera, Bajpai, Terskikh, & Oshima, ). Relevant to the latter point, the fluorescent or tagged antibodies that are currently used for prospective isolation can drive internalization of surface receptors (Liao‐Chan et al, ).…”

DNA‐based probes for flow cytometry analysis of endocytosis and recycling

“…Potential antibody cross‐reactivity is another consideration. Currently, while this approach can be applied for Alexa Fluor‐488 and Alexa Fluor‐594, attempts to generate similar antibodies for Alexa Fluor‐647 and Alexa Fluor‐750 dyes have been unsuccessful …”

“…14,15 Finally, a promising new method that directly measures protein internalization is the use of an anti-flourochrome antibody to quench surface-bound fluorochromes. This approach has been used to measure the internalisation 8,16,17 and recycling 8 of several molecules. While useful, antibodymediated quenching can vary in its efficiency, with some analyses requiring additional controls to ensure adequate exclusion of surface signals.…”

“…Currently, while this approach can be applied for Alexa Fluor-488 and Alexa Fluor-594, attempts to generate similar antibodies for Alexa Fluor-647 and Alexa Fluor-750 dyes have been unsuccessful. 17 We have established an endocytosis assay that overcomes the limitations of other flow cytometry-based assays by using a specific hybridization internalization probe (SHIP, Figure 1A). 18 The SHIP assay comprises of 2 components: a short fluorophore-labeled single-stranded DNA oligonucleotide probe termed fluorescent internalization probe (FIP) and a complementary DNA strand that is conjugated to a quencher dye (QP c ).…”