title: 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.
authors.affiliation: Department of Discovery Research, Igenica Biotherapeutics, Burlingame, California, United States of America.
authors.forename: Sindy
authors.initials: S
authors.lastname: Liao-Chan
authors.affiliation: Department of Discovery Research, Igenica Biotherapeutics, Burlingame, California, United States of America.
authors.forename: Barbara
authors.initials: B
authors.lastname: Daine-Matsuoka
authors.affiliation: Department of Discovery Research, Igenica Biotherapeutics, Burlingame, California, United States of America.
authors.forename: Nathan
authors.initials: N
authors.lastname: Heald
authors.affiliation: Department of Discovery Research, Igenica Biotherapeutics, Burlingame, California, United States of America.
authors.forename: Tiffany
authors.initials: T
authors.lastname: Wong
authors.affiliation: Department of Discovery Research, Igenica Biotherapeutics, Burlingame, California, United States of America.
authors.forename: Tracey
authors.initials: T
authors.lastname: Lin
authors.affiliation: Department of Discovery Research, Igenica Biotherapeutics, Burlingame, California, United States of America.
authors.forename: Allen G
authors.initials: AG
authors.lastname: Cai
authors.affiliation: Department of Discovery Research, Igenica Biotherapeutics, Burlingame, California, United States of America.
authors.forename: Michelle
authors.initials: M
authors.lastname: Lai
authors.affiliation: Department of Discovery Research, Igenica Biotherapeutics, Burlingame, California, United States of America.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: D'Alessio
authors.affiliation: Department of Discovery Research, Igenica Biotherapeutics, Burlingame, California, United States of America.
authors.forename: Jan-Willem
authors.initials: JW
authors.lastname: Theunissen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D000954:Antigens, Surface / Q000276:immunology; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008301:Maleimides / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D021381:Protein Transport; D036104:Receptor, EphA2 / Q000276:immunology; D013050:Spectrometry, Fluorescence
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D005456:Fluorescent Dyes; D008301:Maleimides; C043592:maleimide; D036104:Receptor, EphA2