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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

Variable region gene usage and CDRs for the anti-Alexa Fluor mAbs.The VH and VK genes and heavy and light chain complementarity determining regions (CDRs) for 3 anti-A488 and 3 anti-A594 mAbs are listed. The complete variable region sequences are presented in the S1 Table.
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pone.0124708.g002: Variable region gene usage and CDRs for the anti-Alexa Fluor mAbs.The VH and VK genes and heavy and light chain complementarity determining regions (CDRs) for 3 anti-A488 and 3 anti-A594 mAbs are listed. The complete variable region sequences are presented in the S1 Table.

Mentions: Antibody sequencing was conducted to evaluate anti-Alexa Fluor mAb diversity and to produce the mAbs recombinantly. None of the complementarity determining regions (CDRs) of the anti-A488 mAbs 15A, 19A and 30A shared more than 80% sequence identity (Fig 2 and S1 Table). Tyrosine, a residue previously implicated in fluorescence quenching [19], was present four to five times in the third CDR of the heavy chain of all anti-A488 mAbs. The anti-A594 mAbs shared at least 80% sequence identity in CDR-H3 and all light chain CDRs (Fig 2 and S1 Table).


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)

Variable region gene usage and CDRs for the anti-Alexa Fluor mAbs.The VH and VK genes and heavy and light chain complementarity determining regions (CDRs) for 3 anti-A488 and 3 anti-A594 mAbs are listed. The complete variable region sequences are presented in the S1 Table.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124708.g002: Variable region gene usage and CDRs for the anti-Alexa Fluor mAbs.The VH and VK genes and heavy and light chain complementarity determining regions (CDRs) for 3 anti-A488 and 3 anti-A594 mAbs are listed. The complete variable region sequences are presented in the S1 Table.
Mentions: Antibody sequencing was conducted to evaluate anti-Alexa Fluor mAb diversity and to produce the mAbs recombinantly. None of the complementarity determining regions (CDRs) of the anti-A488 mAbs 15A, 19A and 30A shared more than 80% sequence identity (Fig 2 and S1 Table). Tyrosine, a residue previously implicated in fluorescence quenching [19], was present four to five times in the third CDR of the heavy chain of all anti-A488 mAbs. The anti-A594 mAbs shared at least 80% sequence identity in CDR-H3 and all light chain CDRs (Fig 2 and S1 Table).

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