Transfer of DNA into lymphoma cells by DNA-bound to T101-biotinylated-avidin-polylysine antibody complex
Journal Article
·
· Journal of Nuclear Medicine
OSTI ID:447758
Monoclonal antibodies (MoAb) can be used to target specific cell types. MoAb have been used to selectively deliver DNA molecules to target cells. Our long term goal is to evaluate the use of rapidly internalizing antibody to selectively deliver radiolabeled DNA to the nucleus. As a model system we used T101, an IgG2a murine MoAb that recognizes CD5 and upon binding to this antigen rapidly internalizes. As a DNA sequence we used the pGL2 plasmid containing the luciferase reported gene. Avidin-polylysine was prepared by periodate oxidation of carbohydrate chains of avidin which were then covalently attached to polylysine (Mol.wt 56 kD) and stabilized by sodium borohydride reduction. This conjugate was bound with biotinylated T101 MoAb (biotinylation in lysine residue of MoAb). The final avidin-polylysine-biotin-T101 was purified by size exclusion HPLC. The luciferase DNA was cut by Xba1 restriction enzyme followed by labeling with I-125-dCTP and purified through a G-50 minicolumn. Specific activity was found to be 1{mu}Ci/{mu}g. The I-125 pGL2 was incubated with the T101-biotin-avidin-polylysine complex and 95% of DNA was complexed as determined by nitrocellulose binding. The immunoreactivity of the resulting I-125 pGL2 complex was {approximately} 73%. The retention of I-125 by CD5 positive cells in culture at 37{degrees}C was longer than that of directly iodinated T101. For DNA transfection study CCRF-CEM cell were incubated with 25{mu}G T101-biotin conjugated with avidin-polylysine (10{mu}g) and pGL2 plasmid DNA(10{mu}g). Luciferase activity was observed in the CCRF-CEM cell line that was 35 times higher than the control experiments with pGL2-avidin-polylysine in absence of T101-biotin. In conclusion, these result suggest that DNA conjugated antibody complex can be used to deliver radioactive DNA to the cell nucleus.
- OSTI ID:
- 447758
- Report Number(s):
- CONF-960659--
- Journal Information:
- Journal of Nuclear Medicine, Journal Name: Journal of Nuclear Medicine Journal Issue: Suppl.5 Vol. 37; ISSN 0161-5505; ISSN JNMEAQ
- Country of Publication:
- United States
- Language:
- English
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