Irdye800cw n hydroxysuccinimide ester

Manufactured by LI COR

Sourced in United States

IRDye800CW-N-hydroxysuccinimide ester is a fluorescent dye used for labeling proteins and other biomolecules. It has an absorption maximum at 778 nm and an emission maximum at 806 nm, making it suitable for near-infrared fluorescence applications.

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Lab products found in correlation

Irdye 800cw, by LI COR (2 mentions) Spin desalting column, by Thermo Fisher Scientific (2 mentions) Panitumumab, by Amgen (2 mentions) Control igg, by Innovative Research (1 mentions) Vectibix, by Amgen (1 mentions) Panitumumab, by LI COR (1 mentions) Cetuximab, by LI COR (1 mentions)

5 protocols using irdye800cw n hydroxysuccinimide ester

Labeling Antibodies with IRDye800CW

Cited 2 times

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IRDye800CW (IRDye800CW-N-hydroxysuccinimide ester, LI-COR Biosciences, Lincoln, Nebraska) is nontoxic to mice.^{41 (link)} IRDye800CW is a near infrared imaging probe with a broad absorption (778 nm) and emission (794 nm) peaks. Antibody conjugation results in an absorption of 774 nm and an emission of 789 nm.^{42 ,43 (link)} Control IgG (Innovative Research, Peary Court Novi, MI) and panitumumab (Vectibix; Amgen, Thousands Oaks, California) were labeled according to the manufacturer’s instructions. Briefly, antibodies were incubated with IRDye800CW in 1.00 M potassium phosphate buffer (pH 9.0) for 2 hrs at room temperature. Desalting spin columns (Pierce Biotechnology, Rockford, IL) removed the unconjugated dye. A final dye:protein ratio of 1.5–2.0 was determined using spectrophotometry.

Sweeny L., Prince A., Patel N., Moore L.S., Rosenthal E.L., Hughley B.B, & Warram J.M. (2016). Antiangiogenic Antibody Improves Melanoma Detection by Fluorescently Labeled Therapeutic Antibodies. The Laryngoscope, 126(12), E387-E395.

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Panitumumab-IRDye800 Conjugation and Validation

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Panitumumab (Vectibix; 147kDa, Amgen, Thousand Oaks, CA, USA) and IRDye800CW-N-hydroxysuccinimide ester (1kDa; LI-COR Biosciences, Lincoln, Nebraska, United States) were incubated for 2 h at 20 °C in the dark 30 (link). The dye-to-protein ratio of Panitumumab-IRDye800 was on average 2.0 (range: 1.0 - 3.0) determined by absorption spectrometry (A₇₈₀ / A₂₈₀). Quality control of the conjugate included purity (99.0%), concentration (5.0 mg/mL), excitation/emission peaks (774 nm / 789 nm), EGF receptor binding activity (78.5% relative to unconjugated Panitumumab), pH (7.4) and sterility (no growth). The drug was produced following current Good Manufacturing Practices by the Frederick National Laboratory (Frederick, MD, USA), as part of the National Cancer Institute's NExT Program, and transported in sterile vials to Stanford University, Stanford, CA, USA, under temperature-controlled conditions, and vials were stored and dispensed by the Stanford University Medical Center Investigational Pharmacy.

Zhou Q., van den Berg N.S., Rosenthal E.L., Iv M., Zhang M., Vega Leonel J.C., Walters S., Nishio N., Granucci M., Raymundo R., Yi G., Vogel H., Cayrol R., Lee Y.J., Lu G., Hom M., Kang W., Hayden Gephart M., Recht L., Nagpal S., Thomas R., Patel C., Grant G.A, & Li G. (2021). EGFR-targeted intraoperative fluorescence imaging detects high-grade glioma with panitumumab-IRDye800 in a phase 1 clinical trial. Theranostics, 11(15), 7130-7143.

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Panitumumab-IRDye800 GMP Production

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Panitumumab-IRDye800 was produced under GMP at the Leidos Biomedical Research Center (Frederick, MD). IRDye800CW-NHS (IRDye800CW-N-hydroxysuccinimide ester; LI-COR Biosciences) as a fluorescent probe with a NIR absorption and emission peak of 778 nm/794 nm as discussed previously (17 (link),20 (link),21 (link)). Briefly, panitumumab (Vectibix; Amgen, Thousand Oaks, CA, USA; 147 kDa) was conjugated to IRDye800CW-NHS by a 2-hour incubation at 20°C in the dark with a dye to protein ratio of 2.3:1. Quality control of the conjugate included analysis of drug product in sterile vial for particulates, and integrity of the sterilizing filter. Upon production and vialing, vials were transported to Stanford University where they were stored at the Stanford Health Care Investigational Pharmacy.

Gao R.W., Teraphongphom N.T., van den Berg N.S., Martin B.A., Oberhelman N.J., Divi V., Kaplan M.J., Hong S.S., Lu G., Ertsey R., Tummers W., Gomez A.J., Holsinger F.C., Kong C.S., Colevas A.D., Warram J.M, & Rosenthal E.L. (2018). Determination of Tumor Margins with Surgical Specimen Mapping Using Near-Infrared Fluorescence. Cancer research, 78(17), 5144-5154.

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Covalent Conjugation of Panitumumab with IRDye800CW

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Conjugation of Panitumumab-IRDye800CW was performed under cGMP conditions, as described previously [3 (link)]. Briefly, Panitumumab (Vectibix; Amgen, Thousand Oaks, California, United States) was concentrated, and pH adjusted by buffer exchange to a 10 mg/mL solution in 50 mmol/L potassium phosphate, pH 8.5. IRDye800CW (IRDye800CW-N-hydroxysuccinimide ester, LI-COR Biosciences, Lincoln, Nebraska, United States) was conjugated to Panitumumab for 2 hours at 20°C in the dark, at a molar ratio of 2.3:1. After filtration with desalting columns (Pierce Biotechnology, Rockford, Illinois, United States) to remove unconjugated dye and buffer exchange to PBS, pH 7, the final protein concentration was adjusted to 2 mg/ml. The product was sterilized by filtration and placed into single-use vials and stored at 4°C until used.

Pal R., Hom M., van den Berg N.S., Lwin T.M., Lee Y.J., Prilutskiy A., Faquin W., Yang E., Saladi S.V., Varvares M.A., Rosenthal E.L, & Kumar A.T. (2022). First clinical results of fluorescence lifetime-enhanced tumor imaging using receptor targeted fluorescent probes. Clinical cancer research : an official journal of the American Association for Cancer Research, 28(11), 2373-2384.

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Cetuximab-IRDye800CW Conjugation Protocol

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Anti-EGFR antibody, cetuximab, (Erbitux, ImClone, New York, NY; 152 kDa) was supplied at 2mg/mL. The fluorescent probe, IRDye800CW, (IRDye800CW-N-hydroxysuccinimide ester; LI-COR Biosciences, Lincoln, Nebraska) was supplied as a GMP-compliant reagent. The University of Alabama at Birmingham Vector Production Facility performed the conjugation reaction to prepare the cetuximab-IRDye800CW. Antibodies were incubated with IRDye800CW in 1.00M potassium phosphate buffer (pH 9.0) for 2-hours at room temperature. Desalting spin columns (Pierce Biotechnology, Rockford, IL) removed the unconjugated dye. A final dye:protein ratio of 1.5–2.0 was determined using spectrophotometry.
To produce lower concentrations of cetuximab-IRDye800CW, stock agent (2mg/mL) was diluted in 1X PBS (21-040-CV, Mediatech, Inc., Manassas, VA).

Prince A.C., Jani A., Korb M., Tipirneni K.E., Kasten B.B., Rosenthal E.L, & Warram J.M. (2017). Characterizing the Detection Threshold for Optical Imaging in Surgical Oncology. Journal of surgical oncology, 116(7), 898-906.

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