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41849125559
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note
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+]: 1026.63786. Found 1026.63822.
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21
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41849131370
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note
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+]: 744.30922. Found 744.30692.
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23
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41849105298
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note
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Thiolation of and conjugation with trastuzumab: Trastuzumab (∼7.7 mg) was reacted with Traut's reagent at a 1:15 molar ratio for 1 h at rt in 1 mL of PBS containing 5 mM EDTA buffer. Excess Traut's reagent was removed by passage of the reaction solution through a PD-10 column eluted with PBS containing 5 mM EDTA buffer. Just prior to protein conjugation, 5 was dissolved in the same buffer and then added drop wise to the mAb solution to achieve a molar reaction ratio of 10:1 (5:trastuzumab) and gently vortexed. The solution was then gently agitated in the dark at 25 °C for 1 h. Excess free unreacted SH groups were capped by the addition of iodoacetamide solution (2.0 mM). Finally, the reaction mixture was dialyzed into PBS buffer at 4 °C with 4 buffer changes (4× 1 L) over 48 h to afford immunoconjugate 2. Protein concentration and the number of the CHX-A″ chelate per protein were determined by Lowry assay and Arsenazo(III) assay, respectively. The purity of immunoconjugate 2 was evaluated by both SE-HPLC and SDS-PAGE (data not shown), and was found to be comparable to native trastuzumab.
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25
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41849136778
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111In labeled product was purified on a PD-10 column using PBS as the eluent. The radiolabeling efficiency was 95.2%.
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26
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41849141874
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6/50 μL) in 50 μL of PBS/BSA. The cells were washed with 3 mL of 1% BSA in PBS following an 18 h incubation at room temperature, pelleted at 1000g for 5 min and the supernatant decanted. The radioactivity was measured in a γ-counter (WizardOne, Perkin-Elmer, Shelton, CT) and the percent bound calculated for each dilution. The value presented is the average percent bound.
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