Radiolabelled antibodies

Fig. 7. General scheme for radiolabeling antibodies with rhenium using N2S2-ligand (N3S-ligand)...

Roberts, J.C., Adams, Y.E., Tomalia, D., Mercer-Smith, J.A., and Lavallee, D.K. (1990) Using starburst dendrimers as linker molecules to radiolabel antibodies. Bioconjugate Chem. 1(5), 305-308. 

Subramanian, R., and Meares, C.F. (1991) Bifunctional chelating agents for radiometal-labeled monoclonal antibodies. In Cancer Imaging with Radiolabeled Antibodies (D.M. Coldenberg, ed.), pp. 183-199. Kluwer, Boston, MA. 

Goldenberg, D. 2002. Targeted therapy of cancer with radiolabeled antibodies. Journal of Nuclear Medicine 43(5), 693-713. 

Goldenberg, D. M. (1991) Imaging and therapy of gastrointestinal cancers with radiolabeled antibodies. Am. J. Gastroenterology 86, 1392-1403. 

I-labeled secondary antibody. Secondaiy antibodies can be labeled as previously described (11) or purchased commercially. Radiolabeled antibodies should only be used by personnel trained to properly handle radioisotopes properly... 

Incubate with radiolabeled antibody. Wash to remove unbound antibody. Carry out autoradiography... 

Fig. 2. Western blotting using a radiolabeled antibody (a) SDS polyacrylamide gel electrophoresis, yielding polypeptides separated into discrete bands (b) nitrocellulose or nylon membrane onto which the protein bands have been blotted (i.e. a Western blot) (c) autoradiograph after incubating the Western blot with radiolabeled antibody, washing away unbound antibody and placing the membrane against X-ray film.

Radiolabeled antibody is diluted in BM to an appropriate concentration. Preliminary experiments should be done to determine the binding kinetics and antibody avidity. Routinely we use antibodies at the Kd concentration to give approx half Bmax epitope occupation. 

The protocol can be adapted to measure receptor recycling. If radiolabeled antibodies are internalized into cells by incubation at 37°C in the presence of chemokine or other agents, the acid-elution procedure can be used to remove radiolabeled antibodies remaining on the cell surface. If the acid-elution medium is not reduced below pH 3.0, and the washes kept brief and performed at 4°C, then the cells can be returned to 37°C culture (care should be taken to ensure that the endocytic-trafficking properties of cells are not perturbed by the low pH treatment). During a subsequent incubation at 37°C, receptors that recycle will return antibodies to the cell surface. These antibody molecules can be assessed by measuring the radioactivity that becomes accessible to a second round of acid elution. 

---