Journal: Science Advances

Article Title: Contrasting temporal dynamics of fluorescence and photoacoustic signals from Cetuximab-IRDye800 conjugate in EGFR-overexpressing tumors

doi: 10.1126/sciadv.adv4639

Figure Lengend Snippet: ( A ) Schematic representation of the synthesis of Cetuximab-IRDye800 conjugate. The N -hydroxysuccinimide (NHS) ester of the dye was reacted with the anti-EGFR antibody, Cetuximab, in predefined ratios to develop the conjugate, followed by purification through gel filtration chromatography. “ x ” represents the respective number of dye molecules per antibody. ( B ) Ultraviolet-visible (UV-Vis) absorption spectra of IRDye800 (magenta) and Cetuximab-IRDye800 (cyan) in phosphate-buffered saline (PBS). The spectrum for Cetuximab-IRDye800 shows characteristic absorption peaks for both IRDye800 and Cetuximab along with a peak at 703 nm characteristic of H-aggregate formation. ( C ) SDS-PAGE image of Cetuximab-IRDye800 showing binding of the dye to both the heavy and light chains of the Cetuximab antibody. Free dye, pseudocolored in blue (IRDye800) band, is shown as a reference. HC, heavy chain; LC, light chain. ( D ) Table showing the conjugation ratio of IRDye800 per mole Cetuximab in a reaction to generate Cetuximab-IRDye800 with varying IRDye800 ratios, as mentioned in the sample name. The conjugation ratios for IRDye800 are listed along with the conjugation efficiency. Data are presented as mean ± SD. n ≥ 2 per sample.

Article Snippet: The human anti-EGFR antibody (Cetuximab, ERBITUX from Eli Lilly) was obtained from Premium Health Services (Columbia, MD, USA).

Techniques: Purification, Filtration, Chromatography, Saline, SDS Page, Binding Assay, Conjugation Assay

Journal: Science Advances

Article Title: Contrasting temporal dynamics of fluorescence and photoacoustic signals from Cetuximab-IRDye800 conjugate in EGFR-overexpressing tumors

doi: 10.1126/sciadv.adv4639

Figure Lengend Snippet: ( A ) Hematoxylin and eosin (H&E) and corresponding immunofluorescence image of tumor cross section obtained from a mouse tumor 6 hours after Cetuximab-IRDye-AF injection. ( B ) H&E and corresponding immunofluorescence image of tumor cross section obtained from a mouse tumor 24 hours after Cetuximab-IRDye-AF injection [scale bars, 2.5 mm and 100 μm (inset)]. ( C to G ) Quantification of Cetuximab-IRDye-AF distribution heterogeneity parameters including mean Cetuximab-IRDye-AF intensity (C), %Cetuximab-IRDye-AF coverage area (D), Cetuximab-IRDye-AF–to–CD31 ratio to quantify the amount of Cetuximab-IRDye-AF per blood vessel (E), distance of Cetuximab-IRDye-AF from nearest CD31 + blood vessel (F), and Cetuximab-IRDye-AF–to–EGFR ratio to quantify number of EGFR bound by Cetuximab-IRDye-AF (G). Data are presented as mean ± SD ( n ≥ 10 tumor sections per group from more than five mice each for the 6 and 24 hours and two mice from the untreated group), analyzed using Kruskal-Wallis test, followed by post hoc pairwise comparisons using Dunn’s test, except for Cetuximab-IRDye-AF distance to CD31 (F), which is analyzed using Welch’s t test. P values are provided for each graph.

Article Snippet: The human anti-EGFR antibody (Cetuximab, ERBITUX from Eli Lilly) was obtained from Premium Health Services (Columbia, MD, USA).

Techniques: Immunofluorescence, Injection