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Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

Liao-Chan S, Daine-Matsuoka B, Heald N, Wong T, Lin T, Cai AG, Lai M, D'Alessio JA, Theunissen JW - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Discovery Research, Igenica Biotherapeutics, Burlingame, California, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Quenching by anti-Alexa Fluor mAbs.(A) Fluorescence of Alexa Fluor 488 (A488) on microbeads coated with 1C1-A488 or PC-3 cells stained with 1C1-A488 was quenched with a titration of the benchmark, a rabbit anti-A488 polyclonal, or 1 of 3 anti-A488 mAbs. One representative experiment of multiple is shown. (B) Fluorescence of Alexa Fluor 594 (A594) on microbeads coated with 1C1-A594 or PC-3 cells stained with 1C1-A594 was quenched with a titration of 1 of 3 anti-A594 mAbs. One representative experiment of multiple is shown. (A, B) Median fluorescence intensities (MFIs) at each anti-A488 or anti-A594 mAb concentration were normalized against a buffer control. The chimeric IgG1 isotype control was used as a non-quenching mAb control. The IC50 values (microgram/ml) of quenching and the corresponding 95% confidence intervals (95% CI) are listed for both the microbead- and cell-based titrations.
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pone.0124708.g001: Quenching by anti-Alexa Fluor mAbs.(A) Fluorescence of Alexa Fluor 488 (A488) on microbeads coated with 1C1-A488 or PC-3 cells stained with 1C1-A488 was quenched with a titration of the benchmark, a rabbit anti-A488 polyclonal, or 1 of 3 anti-A488 mAbs. One representative experiment of multiple is shown. (B) Fluorescence of Alexa Fluor 594 (A594) on microbeads coated with 1C1-A594 or PC-3 cells stained with 1C1-A594 was quenched with a titration of 1 of 3 anti-A594 mAbs. One representative experiment of multiple is shown. (A, B) Median fluorescence intensities (MFIs) at each anti-A488 or anti-A594 mAb concentration were normalized against a buffer control. The chimeric IgG1 isotype control was used as a non-quenching mAb control. The IC50 values (microgram/ml) of quenching and the corresponding 95% confidence intervals (95% CI) are listed for both the microbead- and cell-based titrations.

Mentions: PC-3 cells or anti-human IgG microbeads (Miltenyi) were labeled on ice with 100 nM of anti-human EphA2 antibody, a concentration at which staining is saturated (S1 Fig). After a wash step, the cells or beads were incubated for 30 min with buffer or a 6-point 1:4 dilution series of anti-Alexa Fluor mAb starting at 50 microgram/ml. At 10 microgram/ml of anti-Alexa Fluor mAb or a higher concentration, quenching is saturated for 1C1 conjugates (Fig 1) and 3035 conjugates. After another wash step and addition of DAPI, 20,000 live cells or beads were acquired on a MACS-Quant VYB. The median fluorescence intensity (MFI) at each anti-Alexa Fluor mAb concentration was normalized against the buffer control to obtain a normalized MFI percentage. Half maximal inhibitory concentration (IC50) values were derived by using the log(inhibitor) versus response equation in Prism. The IC50 values and their 95% confidence intervals are reported in Fig 1.


Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

Liao-Chan S, Daine-Matsuoka B, Heald N, Wong T, Lin T, Cai AG, Lai M, D'Alessio JA, Theunissen JW - PLoS ONE (2015)

Quenching by anti-Alexa Fluor mAbs.(A) Fluorescence of Alexa Fluor 488 (A488) on microbeads coated with 1C1-A488 or PC-3 cells stained with 1C1-A488 was quenched with a titration of the benchmark, a rabbit anti-A488 polyclonal, or 1 of 3 anti-A488 mAbs. One representative experiment of multiple is shown. (B) Fluorescence of Alexa Fluor 594 (A594) on microbeads coated with 1C1-A594 or PC-3 cells stained with 1C1-A594 was quenched with a titration of 1 of 3 anti-A594 mAbs. One representative experiment of multiple is shown. (A, B) Median fluorescence intensities (MFIs) at each anti-A488 or anti-A594 mAb concentration were normalized against a buffer control. The chimeric IgG1 isotype control was used as a non-quenching mAb control. The IC50 values (microgram/ml) of quenching and the corresponding 95% confidence intervals (95% CI) are listed for both the microbead- and cell-based titrations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403856&req=5

pone.0124708.g001: Quenching by anti-Alexa Fluor mAbs.(A) Fluorescence of Alexa Fluor 488 (A488) on microbeads coated with 1C1-A488 or PC-3 cells stained with 1C1-A488 was quenched with a titration of the benchmark, a rabbit anti-A488 polyclonal, or 1 of 3 anti-A488 mAbs. One representative experiment of multiple is shown. (B) Fluorescence of Alexa Fluor 594 (A594) on microbeads coated with 1C1-A594 or PC-3 cells stained with 1C1-A594 was quenched with a titration of 1 of 3 anti-A594 mAbs. One representative experiment of multiple is shown. (A, B) Median fluorescence intensities (MFIs) at each anti-A488 or anti-A594 mAb concentration were normalized against a buffer control. The chimeric IgG1 isotype control was used as a non-quenching mAb control. The IC50 values (microgram/ml) of quenching and the corresponding 95% confidence intervals (95% CI) are listed for both the microbead- and cell-based titrations.
Mentions: PC-3 cells or anti-human IgG microbeads (Miltenyi) were labeled on ice with 100 nM of anti-human EphA2 antibody, a concentration at which staining is saturated (S1 Fig). After a wash step, the cells or beads were incubated for 30 min with buffer or a 6-point 1:4 dilution series of anti-Alexa Fluor mAb starting at 50 microgram/ml. At 10 microgram/ml of anti-Alexa Fluor mAb or a higher concentration, quenching is saturated for 1C1 conjugates (Fig 1) and 3035 conjugates. After another wash step and addition of DAPI, 20,000 live cells or beads were acquired on a MACS-Quant VYB. The median fluorescence intensity (MFI) at each anti-Alexa Fluor mAb concentration was normalized against the buffer control to obtain a normalized MFI percentage. Half maximal inhibitory concentration (IC50) values were derived by using the log(inhibitor) versus response equation in Prism. The IC50 values and their 95% confidence intervals are reported in Fig 1.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Discovery Research, Igenica Biotherapeutics, Burlingame, California, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus