Limits...
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

Identification of non-competing anti-EphA2 mAbs.(A) Histograms for EphA2-positive cell lines HEC-1-A and PC-3 first stained with unlabeled EphA2 mAb 1C1 or 3035, an IgG control, or buffer only, followed by staining with either labeled 1C1 (1C1-A488) or 3035 (3035-A488). A488 fluorescence is detected by conducting flow cytometry on 10,000 live, single cells. (B) Percent staining by 1C1-A488 or 3035-A488 relative to the HEC-1-A or PC-3 samples first incubated with buffer only.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4403856&req=5

pone.0124708.g003: Identification of non-competing anti-EphA2 mAbs.(A) Histograms for EphA2-positive cell lines HEC-1-A and PC-3 first stained with unlabeled EphA2 mAb 1C1 or 3035, an IgG control, or buffer only, followed by staining with either labeled 1C1 (1C1-A488) or 3035 (3035-A488). A488 fluorescence is detected by conducting flow cytometry on 10,000 live, single cells. (B) Percent staining by 1C1-A488 or 3035-A488 relative to the HEC-1-A or PC-3 samples first incubated with buffer only.

Mentions: The EphA2 receptor tyrosine kinase was one of the targets profiled. After cell binding, the mAb 1C1 induces tyrosine phosphorylation, fast internalization and degradation of the EphA2 receptor [15, 20]. In Fig 3, 1C1 and another anti-EphA2 mAb, 3035, did not compete for binding to EphA2 in two cancer cell lines. In PC-3 cells for example, pre-incubation with 3035 reduced binding of 3035-A488 by 84% relative to the no antibody control (Fig 3B). On the other hand, 3035 had no effect on binding of 1C1-A488, because 1C1 binding was equivalent in the absence or presence of 3035 (Fig 3B), indicating that these two mAbs have non-overlapping epitopes. Previously, it was shown that 1C1 binds an epitope distinct from that of two other anti-EphA2 mAbs [15].


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)

Identification of non-competing anti-EphA2 mAbs.(A) Histograms for EphA2-positive cell lines HEC-1-A and PC-3 first stained with unlabeled EphA2 mAb 1C1 or 3035, an IgG control, or buffer only, followed by staining with either labeled 1C1 (1C1-A488) or 3035 (3035-A488). A488 fluorescence is detected by conducting flow cytometry on 10,000 live, single cells. (B) Percent staining by 1C1-A488 or 3035-A488 relative to the HEC-1-A or PC-3 samples first incubated with buffer only.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124708.g003: Identification of non-competing anti-EphA2 mAbs.(A) Histograms for EphA2-positive cell lines HEC-1-A and PC-3 first stained with unlabeled EphA2 mAb 1C1 or 3035, an IgG control, or buffer only, followed by staining with either labeled 1C1 (1C1-A488) or 3035 (3035-A488). A488 fluorescence is detected by conducting flow cytometry on 10,000 live, single cells. (B) Percent staining by 1C1-A488 or 3035-A488 relative to the HEC-1-A or PC-3 samples first incubated with buffer only.
Mentions: The EphA2 receptor tyrosine kinase was one of the targets profiled. After cell binding, the mAb 1C1 induces tyrosine phosphorylation, fast internalization and degradation of the EphA2 receptor [15, 20]. In Fig 3, 1C1 and another anti-EphA2 mAb, 3035, did not compete for binding to EphA2 in two cancer cell lines. In PC-3 cells for example, pre-incubation with 3035 reduced binding of 3035-A488 by 84% relative to the no antibody control (Fig 3B). On the other hand, 3035 had no effect on binding of 1C1-A488, because 1C1 binding was equivalent in the absence or presence of 3035 (Fig 3B), indicating that these two mAbs have non-overlapping epitopes. Previously, it was shown that 1C1 binds an epitope distinct from that of two other anti-EphA2 mAbs [15].

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