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Ir700 nhs ester

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The IR700 NHS ester is a near-infrared fluorescent dye used for protein labeling. It has an absorption maximum at 689 nm and an emission maximum at 707 nm, making it suitable for detection in the near-infrared range. The NHS ester moiety allows for covalent attachment of the dye to amine groups on proteins or other biomolecules.

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Lab products found in correlation

Coomassie plus protein assay kit, by Thermo Fisher Scientific (15 mentions) Panitumumab, by Amgen (3 mentions) Sephadex g 25 resin, by GE Healthcare (3 mentions) Novaspec plus, by GE Healthcare (2 mentions) Sephadex g50 column pd 10, by GE Healthcare (2 mentions) Sephadex g25 column pd 10, by GE Healthcare (2 mentions) 8453 value system, by Agilent Technologies (2 mentions) Pearl imager, by LI COR (2 mentions) Gradient polyacrylamide gel, by Thermo Fisher Scientific (2 mentions) Clone im7, by BioXCell (2 mentions) Uv 1900, by Shimadzu (1 mentions) Facscalibur, by BD (1 mentions) Cellquest software, by BD (1 mentions) Pm 100, by Thorlabs (1 mentions) Panitumumab, by LI COR (1 mentions) Sephadex g 25 resin, by Cytiva (1 mentions)

15 protocols using ir700 nhs ester

1

Panitumumab and Anti-PD-L1-F(ab')2 Conjugation

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Panitumumab (6.8 nmol) was incubated with IR700 NHS-ester (30.8 nmol, LI-COR Biosciences) in 0.1 mol/L Na2HPO4 (pH 8.6) at 25°C for 1 hour. Anti-PD-L1-F(ab′)2 or control-F(ab′)2 (9.1 nmol) was incubated with IR700 NHS-ester (63.7 nmol, LI-COR Biosciences) in 0.3 mL of 0.1 M Na2HPO4 (pH 8.6) at 25°C for 1 hour.22 (link) The mixture was separated and purified with a Sephadex G50 column (PD-10; GE Healthcare, Piscataway, New Jersey, USA).23 (link) The protein concentration was confirmed with a Coomassie Plus protein assay kit (Thermo Fisher Scientific Inc) by measuring absorption at 595 nm with spectroscopy (UV1900, Shimadzu, Japan).24 25 (link) The IR700 concentration was measured via absorption at 689 nm with spectroscopy to confirm the number of fluorophore molecules conjugated to mAb (dye–mAb ratio).26 (link) Bioactivity of the conjugated products was determined by testing its binding on LL/2-luc-GFP-PD-L1 cells. The cells (1×105) were incubated with pan-700 (10 µg/mL) or anti-PD-L1-F(ab′)2-IR700 (10 µg/mL) in medium for 6 hours at 37°C. For confirming the binding specificity of the new conjugates, a competition assay was performed by adding excess untreated anti-PD-L1 antibody (1 µg). Cells were analyzed with flow cytometry (Gallios, BD Biosciences) using Kalulza2.1 software (BD Biosciences).
Taki S., Matsuoka K., Nishinaga Y., Takahashi K., Yasui H., Koike C., Shimizu M., Sato M, & Sato K. (2021). Spatiotemporal depletion of tumor-associated immune checkpoint PD-L1 with near-infrared photoimmunotherapy promotes antitumor immunity. Journal for Immunotherapy of Cancer, 9(11), e003036.
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2

Synthesis and Characterization of Rova-IR700

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Rovalpituzumab (1 mg, 6.8 nmol) was incubated with IR700 NHS ester (LI-COR Biosciences, Lincoln, NE, USA) (66.8 mg, 34.2 nmol, 5 mmol/L in DMSO) in 0.1 mol/L Na2HPO4 (pH 8.6) at room temperature for 1 h. The mixture was purified using a Sephadex G50 column (PD-10, GE Healthcare; Piscataway, NJ, USA). The protein concentration was determined using a Coomassie Plus protein assay kit (Thermo Fisher Scientific) by spectroscopically measuring the absorption at 595 nm (Novaspec Plus; GE Healthcare). The concentration of IR700 was measured via absorption at 689 nm using spectroscopy to confirm the number of mAb-conjugated fluorophores. The synthesis was controlled so that an average of 3 IR700 molecules were bound to a single antibody; these rovalpituzumab conjugates are referred to as rova-IR700.
Isobe Y., Sato K., Nishinaga Y., Takahashi K., Taki S., Yasui H., Shimizu M., Endo R., Koike C., Kuramoto N., Yukawa H., Nakamura S., Fukui T., Kawaguchi K., Chen-Yoshikawa T.F., Baba Y, & Hasegawa Y. (2020). Near infrared photoimmunotherapy targeting DLL3 for small cell lung cancer. EBioMedicine, 52, 102632.
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3

Protein Conjugation using IR700 NHS Ester

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IR700 NHS ester was obtained from LI-COR Biosciences (Lincoln, NE, USA). Cetuximab was purchased from Bristol-Meyers Squibb (Princeton, NJ, USA). Panitumumab was purchased from Amgen (Thousand Oaks, CA, USA). All other chemicals were of reagent grade.
Okada R., Kato T., Furusawa A., Inagaki F., Wakiyama H., Fujimura D., Okuyama S., Furumoto H., Fukushima H., Choyke P.L, & Kobayashi H. (2022). Selection of antibody and light exposure regimens alters therapeutic effects of EGFRtargeted near-infrared photoimmunotherapy. Cancer immunology, immunotherapy : CII, 71(8), 1877-1887.
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4

Antibody-Fluorophore Conjugation Protocol

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Anti-GPR87 antibody (GPR87ab) (1 mg; 6.8 nmol) was incubated with IR700 NHS ester (LI-COR Biosciences, Lincoln, NE, USA) (66.8 mg; 34.2 nmol, 5 mmol/L in DMSO) in 0.1 mol/L Na2HPO4 (pH 8.6) at 15–25 °C for 1 h. The mixture was purified employing a Sephadex G50 column (PD-10, GE Healthcare; Piscataway, NJ, USA; cat # 17,085,101). The protein concentration was decided employing a Coomassie Plus protein assay kit (Thermo Fisher Scientific; cat # 23,236) by spectroscopically (at 595 nm) (Novaspec Plus; GE Healthcare; cat # 10,187,054). The concentration of IR700 was spectroscopically measured at 689 nm to verify the amount of Ab-conjugated fluorophores [23] (link), [24] (link), [25] (link). The synthesis was controlled in order that a mean of three IR700 molecules were sure to a single antibody; these GPR87ab conjugates with IR700 were abbreviated to as GPR87ab-IR700.
Yasui H., Nishinaga Y., Taki S., Takahashi K., Isobe Y., Shimizu M., Koike C., Taki T., Sakamoto A., Katsumi K., Ishii K, & Sato K. (2021). Near-infrared photoimmunotherapy targeting GPR87: Development of a humanised anti-GPR87 mAb and therapeutic efficacy on a lung cancer mouse model. EBioMedicine, 67, 103372.
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5

Conjugation and Binding of Anti-CD25 F(ab')2

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Anti-CD25-F(ab’)2 or control-F(ab’)2 (9.1 nmol) was incubated with IR700 NHS ester (45.5 nmol, LI-COR Biosciences) in 0.3 ml of 0.1 M Na2HPO4 (pH 8.6) at room temperature for 1 hour. The mixture was purified with a Sephadex G-25 column (PD-10; GE Healthcare). The protein concentration was determined using the Coomassie Plus Protein Assay Kit (Thermo Fisher Scientific Inc.) by measuring the absorption at 595 nm using 8453 Value System (Agilent Technologies). The concentration of IR700 was determined by measuring the absorption at 689 nm, and the number of fluorophore molecules conjugated to each F(ab’)2 molecule was calculated. The conjugation was performed such that an average of three IR700 molecules was bound to a single F(ab’)2. We performed SDS–polyacrylamide gel electrophoresis to confirm the integrity of IR700-conjugated F(ab’)2, and the bioactivity was confirmed by examining its binding to HT-2-A5E cells. The cells (1 × 105) were incubated with anti-CD25-F(ab’)2–IR700 (10 μg/ml) in medium for 1 to 6 hours at 37°C. To validate the specificity of the binding, a competition assay was performed by adding excess untreated anti-CD25 antibody (50 μg). Cells were analyzed by flow cytometry (FACSCalibur, BD Biosciences) using the CellQuest software (BD Biosciences).
Sato K., Sato N., Xu B., Nakamura Y., Nagaya T., Choyke P.L., Hasegawa Y, & Kobayashi H. (2016). Spatially selective depletion of tumor-associated regulatory T cells with near-infrared photoimmunotherapy. Science translational medicine, 8(352), 352ra110.
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6

Synthesis of Anti-CD44-IR700 Antibody

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Anti-mouse/human-CD44 antibody (1 mg, 6.7 nmol; clone IM7; Bio X Cell, West Lebanon, NH, USA) was incubated with IR700 NHS ester (65.1 μg, 33.3 nmol, 10 mmol/L in DMSO; LI-COR Bioscience, Lincoln, NE, USA) and phosphate buffer (pH 8.5; Teknova, Hollister, CA, USA) at room temperature for 1 h. The mixture was purified with a gel filtration column (Sephadex G 25 column, PD-10, GE Healthcare, Piscataway, NJ, USA). Protein concentration was determined with Coomassie Plus protein assay kit (Thermo Fisher Scientific Inc, Rockford, IL, USA) by measurement of the absorption at 595 nm with spectroscopy (8453 Value System; Agilent Technologies, Santa Clara, CA, USA). We abbreviate IR700-conjugated anti-CD44 antibody as anti-CD44-IR700.
Moriya T., Hashimoto M., Matsushita H., Masuyama S., Yoshida R., Okada R., Furusawa A., Fujimura D., Wakiyama H., Kato T., Choyke P.L., Kusumoto Y., Chtanova T., Kobayashi H, & Tomura M. (2022). Near-infrared photoimmunotherapy induced tumor cell death enhances tumor dendritic cell migration. Cancer immunology, immunotherapy : CII, 71(12), 3099-3106.
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7

Anti-GPC3 Mouse Monoclonal Antibodies

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Anti-GPC3 mouse mAb YP7 (IgG) and HN3 (VH-hFc) were developed in house.13 (link),15 (link) A431/G1, a cell line stably expressing human GPC3, was established.15 (link) IR700 NHS ester was obtained from LI-COR Bioscience (Lincoln, NE, USA). Other reagents were of reagent grade and were used as received.
Hanaoka H., Nagaya T., Sato K., Nakamura Y., Watanabe R., Harada T., Gao W., Feng M., Phung Y., Kim I., Paik C.H., Choyke P.L., Ho M, & Kobayashi H. (2015). Glypican-3 Targeted Human Heavy Chain Antibody as a Drug Carrier for Hepatocellular Carcinoma Therapy. Molecular pharmaceutics, 12(6), 2151-2157.
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8

Antibody-Photosensitizer Conjugation Protocol

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One milligram (6.7 nmol) of anti-CD29 and anti-CD44 antibodies (clone KM16 and IM7, respectively, purchased from Bio X Cell) were incubated with 66.8 μg (34.2 nmol) of IR700 NHS ester (Li-Cor) in 0.1 M Na2HPO4 solution (pH 8.5) for 1 hour at room temperature. The mixture was purified using a PD-10 Desalting Column with Sephadex G-25 resin (Cytiva) and eluted with PBS. The resulting AbPC solution was diluted to make 1.675 nmol/mL. AbPCs were analyzed by SDS-PAGE with a 4–20% gradient polyacrylamide gel (Life Technologies). The same amount of unconjugated antibodies were loaded next to the AbPCs as controls. The gel was imaged with a Pearl Imager (LI-COR Biosciences) using a 700 nm fluorescence channel. The gel was stained with Colloidal Blue staining to determine the molecular weight of AbPCs.
Furusawa A., Okada R., Inagaki F., Wakiyama H., Kato T., Furumoto H., Fukushima H., Okuyama S., Choyke P.L, & Kobayashi H. (2022). CD29 targeted near-infrared photoimmunotherapy (NIR-PIT) in the treatment of a pigmented melanoma model. Oncoimmunology, 11(1), 2019922.
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9

Synthesis and Purification of CD44-IR700

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APC was synthesized as described previously (19 (link)). In brief, 1 mg of anti-mouse/human CD44 monoclonal antibody (6.7 nmol; clone IM7; Bio X Cell, West Lebanon, NH, USA) was incubated with five-fold molar excess of IR700 NHS ester (10 mM in DMSO; LI-COR Biosciences, Lincoln, NE, USA) in 100 mM Na2HPO4 solution (pH 8.5) for one hour at room temperature. The mixture was purified with PD-10 columns containing Sephadex G25 resin (GE Healthcare, Piscataway, NJ, USA). The resulting APC was abbreviated as CD44-IR700.
Rothermich M.M., Guerrero R., Lenz R.W, & Goodwin S. (2000). Characterization, Seasonal Occurrence, and Diel Fluctuation of Poly(hydroxyalkanoate) in Photosynthetic Microbial Mats. Applied and Environmental Microbiology, 66(10), 4279-4291.
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10

Antibody-Mediated Photodynamic Therapy

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Anti-GPC3 mouse mAb YP7 was developed in house [15 (link)]. Nanoparticle albumin-bound paclitaxel (nab-paclitaxel; Abraxane®) was purchased from Celgene (Summit, NJ, USA). A431/G1, a cell line stably expressing human GPC3, was established [15 (link)]. IR700 NHS ester was obtained from LI-COR Bioscience (Lincoln, NE, USA). A red light-emitting diode (LED) light source, which emits light at 690 ± 20 nm (antigen wavelength (L690–66–60, Marubeni America Co., CA, USA) was used for NIR light irradiation of PIT in vitro and in vivo. A power density was measured with an optical power meter (PM100, Thorlabs, NJ, USA) and exposure with this LED light for 1 min was calculated as 2.5 J/cm2. Other reagents were of reagent grade and were used as received.
Hanaoka H., Nakajima T., Sato K., Watanabe R., Phung Y., Gao W., Harada T., Kim I., Paik C.H., Choyke P.L., Ho M, & Kobayashi H. (2015). Photoimmunotherapy of hepatocellular carcinoma-targeting Glypican-3 combined with nanosized albumin-bound paclitaxel. Nanomedicine (London, England), 10(7), 1139-1147.
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