Antibody Radiolabeling & Screening

Antibody Research-  Radiolabeling & Screening

One way the immune system attacks foreign substances in the body is by making large numbers of antibodies. An antibody is a protein that sticks to a specific protein called an antigen. Antibodies circulate throughout the body until they find and attach to the antigen. Once attached, they can recruit other parts of the immune system to destroy the cells containing the antigen.

Researchers can design antibodies that specifically target a certain antigen, such as one found on cancer cells. They can then make many copies of that antibody in the lab. These are known as monoclonal antibodies (mAbs).

Over the past couple of decades, the US Food and Drug Administration (FDA) has approved more than a dozen mAbs to treat certain cancers. As researchers have found more antigens linked to cancer, they have been able to make mAbs against more and more cancers.

METIS Labs can label your antibodies with radioiodine using a simple procedure. The labeled primary antibody can then be used to directly measure both antibody affinity (Kd) and the density of antigen sites (sites/cell) in live cells. Radioactive assays often have higher sensitivity, robustness and quantification when compared with comparable fluorescent-based assays.

Antibody (Ab) Radiolabeling

Stock Antibody and Na[125I]Iodide are mixed with IodoGen®. After a set amount of time the mixture is transferred to a fresh vial and the reaction is quenched. The mixture is then purified using a special column and equilibrated with buffer. The [125I]-Ab can be shipped to, or used in house for:

  • i. Saturation Binding Assay
  • ii. Competition Binding Assay
  • iii. Homologous Competition Binding Assay

Antibody (Ab) Saturation Assay (Sites/cell & Kd)

Multiple concentrations (up to ten) of the [125I]-Ab are incubated in triplicate (or quadruplicate) with a desired cell line containing the target of interest.

After equilibrium has been reached, receptor-bound [125I]-Ab is separated from non-bound radioligand using either

i.  Filter-based Separation
ii. Scintillation Proximity Assay (SPA)

The filters or SPA plate are counted using liquid scintillation counting, or Gamma Counting, to determine the level of receptor-bound radioligand and the results plotted to obtain sites/cell and if desired the Kd for the target-radioligand integration.

Antibody (Ab) Competition Binding Assay (IC50 and Ki)

Live cells containing the target of interest are incubated together with a [125I]-Ab at a single concentration along with 8 or 10 concentrations unlabeled Ab in triplicate (or quadruplicate) replicates at each concentration.

After equilibrium has been reached, receptor-bound radioligand is separated from non-bound radioligand using either.

i.  Filter-based Separation
ii. Scintillation Proximity Assay (SPA)

The filters or SPA plates are counted for radioactivity to determine the level of target-bound radioligand and the results are plotted to obtain the IC50 and Ki values for the Ab-target interaction.

Antibody (Ab) Homologous Competition

A homologous competition is a concentration response curve with an unlabeled compound that is identical to the radioligand being used. Radioligand concentration is constant in the experiment. Homologous competition experiments can be used as an alternative to saturation experiments to determine receptor affinity (Kd) and density (Bmax). When using [125I]-ligands, a non-radioactive iodo-ligand should be used if possible.