The humanized M5A mAb was developed by grafting the CDR region of the murine anti-CEA antibody mT84.66 onto the humanized anti-p185HER2 antibody (Trastuzumab) framework and expressed as previously described (16 (link)). The purified hM5A antibody was conjugated with NHS-IRDye800CW (LI-COR Biosciences, Lincoln, NE) at a 10-fold molar excess of the esterified dye at room temperature for 1 hour. Concentrations of the hM5A-IR800 conjugate were determined by Absorbance280. The final concentration of antibody-dye conjugate was 5.7 mg/mL with an average of 2 dye molecules per IgG based on mass spectrometry.
Irdye800cw nhs
Manufactured by LI COR
Sourced in United States
IRDye800CW-NHS is a near-infrared fluorescent dye that can be used for labeling proteins, peptides, and other biomolecules. It has an excitation maximum of 774 nm and an emission maximum of 789 nm, making it suitable for use in a variety of imaging and detection applications.
Automatically generated - may contain errors
Lab products found in correlation
Bevacizumab, by Roche (2 mentions)
Traut s reagent 2 iminothiolane hcl, by Thermo Fisher Scientific (2 mentions)
Scm peg5k mal, by Creative PEGWorks (2 mentions)
Nhs fluorescein, by Merck Group (2 mentions)
Pd 10 column, by GE Healthcare (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Kaiser test kit, by Merck Group (2 mentions)
Nota scn, by Macrocyclics (2 mentions)
Cetuximab, by Merck Group (2 mentions)
Plasmid safe dnase, by Illumina (1 mentions)
Hydroxyethyl starch viastarch hes, by Fresenius (1 mentions)
Ir 780, by Eppendorf (1 mentions)
Ir780, by Merck Group (1 mentions)
Chelex 100 resin, by Bio-Rad (1 mentions)
Pertuzumab, by Genentech (1 mentions)
Desferrioxamine p benzyl isothiocyanate dfo bz ncs, by Macrocyclics (1 mentions)
Lachrom system, by Hitachi (1 mentions)
Trastuzumab, by Genentech (1 mentions)
3 aminopropyl triethoxysilane aps, by Merck Group (1 mentions)
Chelex 100 resin, by Merck Group (1 mentions)
16 protocols using irdye800cw nhs
1
Humanized Anti-CEA Antibody Conjugation
2
Antibody-IRDye800CW Conjugation Protocol
3
Humanized Anti-HER2 Antibody Conjugation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The humanized hT84.66-M5A (hM5A) mAb was established by grafting the CDR region of the murine mT84.66 mAb onto a human anti-p185HER2 antibody (Trastuzumab) framework, as previously described [23 (link)]. Purified hM5A antibody was conjugated with NHS-IRDye800CW (LI-COR Biosciences, Lincoln, NE, USA) at 10-fold molar excess of dye at room temperature for 1 hour. Absorbance at 280 nm was used to determine concentration of the fluorophore-conjugated antibody. Final concentration of antibody-dye conjugate was 5.7 mg/mL. Mass spectrometry was used to determine an average of 6 dye molecules per IgG.
4
Optimized Trehalose Adjuvant Formulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemicals were reagent grade. High purity, low endotoxin α,α-trehalose dihydrate was a generous donation of Pfanstiehl (Waukegan, Illinois). Two percent Alhydrogel® (aluminum hydroxide adjuvant) was obtained from Accurate Chemicals and Scientific Corp (Westbury, NY). 3,3′,5,5′-tetramethylbenzidine (Turbo TMB) and peroxidase-conjugated donkey anti-mouse IgG (H + L) was from Thermo Scientific (Rockford, IL). Plasmid-safe DNase was from Epicentre (Madison, WI). Hydroxyethyl starch/Viastarch (HES) was obtained from Fresenius Kabi, Austria, GmbH. IR Dye 800CW NHS was obtained from LI-COR Biosciences, Bad Homburg, Germany.
5
Bevacizumab-800CW Tracer Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Clinical grade bevacizumab-800CW was produced in the good manufacturing practice (GMP) facility of the UMCG by conjugating bevacizumab (Roche AG) and IRDye-800CW-NHS (LI-COR Biosciences Inc) under regulated conditions16 (link). The average conjugation molecule ratio of bevacizumab (molecular weight: 149 KDa) to IRDye-800CW-NHS (molecular weight: 1.166 KDa) was 1:2, generating the conjugate bevacizumab-800CW with a total molecular weight of 151.3 KDa. Vials containing 6.0 mg bevacizumab-800CW dissolved in 0.9% sodium chloride (NaCl) solution were used to prepare the infusions in a concentration of 1 mg ml−1. After release of the final product by the certified qualified person at the UMCG GMP facility, the tracer was intravenously administered to the subjects.
6
Protein Labeling with IR-780 and IR-800
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The labeling of BSA was taken as an example. BSA [10 μM in phosphate-buffered saline (PBS); Meilunbio, MA0015] was pipetted into an Eppendorf tube, and IR-780 [Sigma-Aldrich, 425311; 2 mM in dimethyl sulfoxide (DMSO)] was added to the Eppendorf tube, ensuring that the molar ratio of IR-780/BSA was 1:1. Then, the solution was placed in a 50°C shaker to react for 2 hours to obtain the dye-labeled protein samples. β-LG, OVA, and Tf were labeled by IR-780 (at 70°, 90°, and 80°C, respectively) following the same protocol. In addition, IRDye800CW-NHS (IR-800; LI-COR Biosciences, 929-70020) was used to label proteins at 50°C (the optimum temperature for binding IR-780 to BSA) following the same method, which was used as a control experiment.
The binding ability of IR-780 to the protein varied with temperature, we thus conducted a series of IR-780 labeling experiments for AKT1 at 30°, 40°, 50°, 60°, 70°, and 80°C, respectively. In detail, AKT1 (2.5 μM in double distilled H2O) was pipetted into an Eppendorf tube, and IR-780 (2 mM in DMSO) was added to the Eppendorf tube, ensuring that the molar ratio of AKT1/IR-780 was 1:1. The solution was placed in a shaker to react at the specified temperature for 2 hours to obtain dye-labeled AKT1 samples.
The binding ability of IR-780 to the protein varied with temperature, we thus conducted a series of IR-780 labeling experiments for AKT1 at 30°, 40°, 50°, 60°, 70°, and 80°C, respectively. In detail, AKT1 (2.5 μM in double distilled H2O) was pipetted into an Eppendorf tube, and IR-780 (2 mM in DMSO) was added to the Eppendorf tube, ensuring that the molar ratio of AKT1/IR-780 was 1:1. The solution was placed in a shaker to react at the specified temperature for 2 hours to obtain dye-labeled AKT1 samples.
7
Radiolabeling of Therapeutic Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All reagents were of analytical grade and used without further purification unless otherwise stated. Chelex-100 resin was purchased from Bio-Rad Laboratories (Richmond, CA) and used with aqueous buffers for radiolabeling experiments to ensure metal-free conditions. Trastuzumab and pertuzumab were obtained from Genentech (San Francisco, CA). Control IgG1 antibodies, 88R20 and mAb-69 were kindly provided by Dr. Kendra Carmon and Dr. Barret R. Harvey, the University of Texas Health Science Center at Houston. IRDye800CW-NHS was purchased from LI-COR Biosciences (Lincoln, NE). Desferrioxamine-p-benzyl-isothiocyanate (DFO-Bz-NCS) was purchased from Macrocyclics (Plano, TX). Zirconium-89 (89Zr)-oxalate was produced by Washington University School of Medicine (St. Louis, MO). Size-exclusion high-performance liquid chromatography (SEC-HPLC) was performed on an analytical Hitachi LaChrom system using a TSKgel G3000SW (5 μm) column and mobile phases of A = 0.1 M sodium phosphate buffer (pH 7.3) and B = CH3CN (isocratic: 90% A and 10% B) and a flow rate of 1 mL/min. Radiochemical yield and purity were measured with a radio-thin-layer chromatography (TLC)/HPLC detector system (LabLogic) using previously described methods [15 (link)].
8
Bioconjugation of TRC105 with IR Dye 800CW
9
Cetuximab Conjugation with IRDye800CW
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cetuximab (Erbitux, Ely Lily; 2 mgml-1, 145781.6 g/mol as per FASTA sequence analysis) was conjugated to IRDye800CW (IRDye800CW-NHS Ester; Li-COR, 1166.20 g/mol) by amide coupling. A stock solution of IRDye800CW-NHS Ester (10 mg·ml-1) was prepared in anhydrous Dimethyl Sulfoxide (DMSO; Sigma-Aldrich) and added to Cetuximab (2 mg·ml-1 in phosphate buffer) at a quantity corresponding to a 10-fold molar excess of IRDye800CW to each molecule of Cetuximab. The mixture was thoroughly mixed using orbital rotation for 24h at room temperature. After conjugation, the Cet-IRDye800 was purified by removing unconjugated dye using PD-10 Desalting Columns (Cytivia) pre-equilibrated with sterile 1X DPBS (Corning). Purified Cet-IRDye800 was collected and concentrated in a 30kDa ultrafiltration tube (EMD Millipore) by centrifugation at 2,500 xg for 20 min at 4°C. The conjugated Cet-IRDye800CW was then stored at 4°C in the dark.
10
Bioconjugation of TRC105 with IR Dye
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRC105 was provided by TRACON Pharmaceuticals
Inc. (San Diego, CA). PD-10 columns were purchased from GE Healthcare
(Piscataway, NJ). IR Dye 800CW-NHS (NHS denotes N-hydroxysuccinimide) ester was acquired from LI-COR Biosciences Co.
(Lincoln, NE). SCM-PEG5k-Mal was obtained from Creative
PEGworks. NOTA-SCN (i.e., 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic
acid) was acquired from Macrocyclics, Inc. (Dallas, TX). NHS-fluorescein,
Chelex 100 resin (50–100 mesh), tetraethyl orthosilicate (TEOS),
ammonia (NH3·H2O), triethylamine (TEA),
3-aminopropyl)triethoxysilane (APS), dimethyl sulfoxide, cetyltrimethylammonium
chloride (CTAC, 25 wt %), and Kaiser test kit were purchased from
Sigma-Aldrich (St. Louis, MO). Traut’s reagent (2-Iminothiolane·HCl)
and Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic
acid) or DTNB) were purchased from Fisher Scientific. Water and all
buffers were of Millipore grade and pretreated with Chelex 100 resin
to ensure that the aqueous solution was free of heavy metals. All
chemicals were used as received without further purification.
Inc. (San Diego, CA). PD-10 columns were purchased from GE Healthcare
(Piscataway, NJ). IR Dye 800CW-NHS (NHS denotes N-hydroxysuccinimide) ester was acquired from LI-COR Biosciences Co.
(Lincoln, NE). SCM-PEG5k-Mal was obtained from Creative
PEGworks. NOTA-SCN (i.e., 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic
acid) was acquired from Macrocyclics, Inc. (Dallas, TX). NHS-fluorescein,
Chelex 100 resin (50–100 mesh), tetraethyl orthosilicate (TEOS),
ammonia (NH3·H2O), triethylamine (TEA),
3-aminopropyl)triethoxysilane (APS), dimethyl sulfoxide, cetyltrimethylammonium
chloride (CTAC, 25 wt %), and Kaiser test kit were purchased from
Sigma-Aldrich (St. Louis, MO). Traut’s reagent (2-Iminothiolane·HCl)
and Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic
acid) or DTNB) were purchased from Fisher Scientific. Water and all
buffers were of Millipore grade and pretreated with Chelex 100 resin
to ensure that the aqueous solution was free of heavy metals. All
chemicals were used as received without further purification.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!