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

Multiple concentrations (up to twelve) of the [125I]-Ab are incubated in 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:

  • Filter-based Separation
  • 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 saturation binding plate set-up in a 96-well plate format. The plate is designed to contain 2 cell lines (Columns 1 - 6 & 7 - 12, each in triplicate). Total binding (column 1 - 3; 7 - 9) and nonspecific binding (column 4 - 6; 10 - 12) are determined in the absence and presence of 10 μM of appropriate reference compound, respectively, in the final concentrations of hot ligand (0.04 - 5 nM; rows A - H).
Antibody saturation binding plate set-up in a 96-well plate format. The plate is designed to contain 2 cell lines (Columns 1 - 6 & 7 - 12, each in triplicate). Total binding (column 1 - 3; 7 - 9) and nonspecific binding (column 4 - 6; 10 - 12) are determined in the absence and presence of 10 μM of appropriate reference compound, respectively, in the final concentrations of hot ligand (0.04 - 5 nM; rows A - H).
Antibody Saturation Binding Data GraphPad
Antibody Saturation Binding Data GraphPad