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To directly radiolabel an anti-hepatoma mAb fragment HAb18 F(ab’)2 with 99mTc …


Biology Articles » Biophysics » Medical Biophysics » Direct technetium-99m labeling of anti-hepatoma monoclonal antibody fragment: a radioimmunoconjugate for hepatocellular carcinoma imaging » Results

Results
- Direct technetium-99m labeling of anti-hepatoma monoclonal antibody fragment: a radioimmunoconjugate for hepatocellular carcinoma imaging

Figure 1 represents the calibration curve for the determination of sulphydryl groups using L-cys standards over a range of 0.312 to 10mg/L, by plotting optical density at 412nm versus L-cys standard concentrations after subtraction of the background due to Ellman’s reagent. Linear regression was used and correlation coefficient 0.999 was obtained. Table 1 shows the influence of the reduction conditions on the number of free sulphydryl groups detected by this thiol assay. As expected, increasing the molar ratio of Sn/GH to antibody in the reaction mixture does increase the number of apparent -SH groups per antibody, and increase the labeling efficiency correspondingly, which results in the labeling efficiency at a maximum of 84.2%. The free 99mTcO4- and colloid amounts determined by Whatman 3MM paper using different developing systems were also showed in Table 1. In control expe ri ments, labeling efficiency was 2% when unreduced HAb18 F(ab’)2 was used. SD S-PAGE by both staining and autoradiography showed that the radioactivity co-m igrated with the proteins and that there were almost no protein fragments prese nt within the 601 of molar ratio of Sn/GH to mAb (Figure 2). However , another SDS-PAGE in Figure 3 illustrates that fragmentation occurred during t he reduction procedure when the molar ratio of Sn/GH to mAb was at 500 1.
      As shown in Figure 4, the immunoreactive fraction, 0.84 was determined by plott ing the inverse of the bound fraction compared with the inverse of the cell conc entration, which is based on the assumption that the total antigen concentration (cell concentration) is a good enough approximation for the free antig en concentration.
      Challenging with EDTA did not remove 99mTc from the labeling conjugat e remarkably, while L-cys at a molar ratio of 6251 remove approximately one-tenth of the label (Figure 5).
      Biodistribution of radioactivity in blood and excised tissues are displayed in Table 2. The preparation localized at the tumors was more than at any organ examined at both 10h and 24h after injection, except the kidneys. Th e lower radioactivity in blood at 24h suggested fast blood clearance. The imaging results in Figure 6 showed significant tumor uptake at 24h post-i njection.

 


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