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

Content Provider	PubMed Central
Author	Liao-chan, Sindy Barbara, Daine-matsuoka Heald, Nathan Wong, Tiffany Lin, Tracey Cai, Allen G. Lai, Michelle D’alessio, Joseph A. Theunissen, Jan-willem
Editor	D'auria, Sabato
Copyright Year	2015
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.
Related Links	http://dx.doi.org/10.1371/journal.pone.0124708
Starting Page	124708
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	4
Volume Number	10
Language	English
Publisher	Public Library of Science
Publisher Date	2015-04-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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

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

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