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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 » Materials and Methods

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

Monoclonal antibody
The mAb HAb18 is of murine IgG1 isotype and was developed by our laboratory[18]. F(ab’)2 fragment of HAb18 was generated by papain digestion with a molecular weight of
96000 dalton[19].

Hepatocellular carcinoma grown in Balb/c mice was used as a prototype tumo r model. Approximately 107 HHCC cells obtained from Shanghai Cell Institute of Chinese Academy of Sciences were implanted in the left thigh of the animals and the tumors were allowed to grow for 8-10 days to approximately 1cm in diameter.

Antibody reduction
The antibody concentrated to 8g/L in neutral PBS was reduced by reaction with a molar excess of stannous/glucoheptonate (Sn/GH) ranging from 10
1 to 501 (Sn/GH: MAb) at 37 for 15min-30min. The Sn/GH with a mass ratio of 1100 was dissolved in 50mM acetate-buffered saline (ABS), pH 5.3 purged with nitrogen. The reduced antibody was isolated from reductant t hro ugh a PD-10 column (Pharmacia) equilibrated with 0.05mol/L ABS. The number of resulting free sulphydryl groups was assayed with Ellman’s reagent 55’dithio-bis (2-nitrobenzoic acid), (DTNB, Sigma Chemical Co., USA) [20]. One hundred μL of sample was mixed with 20μL of 0.01mol/L-DTNB and diluted to 3mL with 0.05mol/L Tris-HCl buffer pH 8.4. The mixture was incubated at room te mperature for 15min and coloration measured with an UV/VIS spectrophotome ter at 412nm. The number of thiols was obtained by comparison with a seri es of L-cysteine (L-cys) standards ranging from 0.312mg/L to 10mg/L.
      The integrity of the reduced F(ab’)2 was determined by non-reduced SDS-PAGE with 100g/L gel using Vertical Gel Electrophoresis System (Bio-Rad). The gel was stained with Coomassie brilliant blue R250.Control experiments were run using unreduced mAb F(ab’)2.

For labeling, 160μg of reduced HAb18 F(ab’)2 was mixed with a 10μL-20μL of diluted Sn/GH solution (0.2g/L), and pertechnetium solution (0.2mL , 74MBq), (Chinese Academy of Atomic Energy) was injected into the mixture . The Sn/GH solution was freshly prepared each time by dissolving 100mg GH and 1mg SnCl2·2H2O in 5mL of saline purged with nitrogen. The reaction mixture was incubated for 0.5h-1h at 37
before it was analyzed by Whatman 3MM paper chromatography which was then developed in acetone or 100g/L trichloroacetic acid (TCA). R-f values for acetone are: mAb 0.0, 99mTc-GH 0.0, and 99mTcO4-0.9-1.0. R-f values for 100g/L TCA are: mAb 0.0, 99mTcGH 0, and 99mTcO4- 0.7. Labeled mAb was differentiated from 99mTc colloid by the method of Thrall et al[21]. The same strips impregnated with 10g/L-20g/L human serum albumin before development with 521, waterethanol5N NH4OH. Colloid remained on the bottom of the strip while mAb-bound isotope migrated with the solvent front.
      The integrity of the labeled F(ab’)2 was assayed using the same non-reduced SDS-PAGE as described above. The gel was autoradiograghied on x-ray film before stained with Coomassie brilliant blue R250.

Immunoreactivity assessment
The in vitro immunoreactivity of the radiolabeled HAb18 F(ab’)2 was evalu ated by a live cell assay[22]. Briefly, HHCC cells 5×109/L were centrifuged (1000r/min) for 5min and washed twice with 1% bovine serum albumin (BSA) in PBS, then 5 serial 1
2 dilutions were made in 10g/L BSA in Eppendorf tubes precoated with BSA. Radiolabeled HAb18 F(ab’)2 at a concentration of 40ng/mL in 10g/L BSA was added using a volume equal to half the volume of cell suspension. The total volume of cell-binding assay solution was 0.3mL. After incubation for 2h at 37, the total as well as the cell-bound radioactivity were counted in a gamma counter.

In Vitro stability studies
The stability was analyzed by using two different challenging agents, EDTA and L-cys. An aliquot of 50μL 99mTc-HAb 18 F(ab’)2 solution was incubated with EDTA or L-cys at 37
for 1h. The molar ratio of mAb to challenging agent was at a maximum of 100001. Dissociation ratio was analyzed on paper chromatography.

Biodistribution and imaging
Balb/c mice bearing HHCC were divided into three groups. Each group consisted of three animals and each animal received approximately 15μ g antibody with about 7.4MBq through a lateral tail vein. At time i ntervals of 4, 10 and 24h postinjection, three groups of mice were killed , and imaged on a SPECT (Starcam 3000, UK). Data were collected 100000 co unts per image and peak energy settings at the 140ke V (20%) window for 99mTc. The blood and other organs of interest were collected. Tissues were w ashed, blotted, weighed and counted in a gamma counter. For each mouse, data are expressed as percent of injected dose per gram of tissue (%ID/g) after physical decay corrected.

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