Journal: Bioconjugate Chemistry

Article Title: Enhancing Reactivity for Bioorthogonal Pretargeting by Unmasking Antibody-Conjugated trans-Cyclooctenes

doi: 10.1021/bc500605g

Figure Lengend Snippet: Figure 5. Translation of dual orthogonal coupling to different antibodies. (A) Anti-TfR and EpCAM antibodies had low TCO reactivity (12−15%) after direct amine-conjugation, but the PEG24− TCO increased reactive TCO density 3- to 4-fold. *MALDI-TOF analysis of the TCO−PEG24-modified anti-EpCAM antibody was limited by difficulties with desorption. (B) Flow cytometry results for TCO- and TCO−PEG24-modified anti-EpCAM and TfR antibodies on A431 cells after reaction with 10 nM tetrazine−QDs for 30 min, showing 5-fold signal enhancements for the PEG24−TCO. (C) Confocal images of A431 cells labeled with (i, iv) anti-EpCAM, (ii, v) anti-TfR, or (iii, vi) nonbinding control antibodies. The antibodies were modified with (i−iii) TCO or (iv−vi) PEG24−TCO at the highest densities and reacted with tetrazine−QD at 10 nM for 30 min. Scale bar: 20 μm. Error bars represent the standard error of at least three independent experiments.

Article Snippet: Monoclonal mouse antibodies specific for human EpCAM (IgG2b, clone 158206) and transferrin receptor (TfR, IgG1, clone 29806) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Conjugation Assay, Flow Cytometry, Labeling, Control