Section5:Assay Conditions (Filter)

From Assay Guidance Wiki

Incubation Time - Signal Stability

Setup: Measure total binding (receptor + radioligand) and nonspecific binding (receptor + radioligand + excess unlabeled competitor) at various times.

At least two methods could be used to obtain the association/dissociation data. Both methods should yield the same result. If not, there may be a problem with receptor stability.

Method 1: Add and mix together enough receptor and radioligand for all time points in the experiment. At various times, filter an aliquot of the receptor/radioligand mixture and wash the filters with Wash Buffer. The last aliquots to be filtered will be the longest incubation time points.

Method 2: Prepare receptor and radioligand separately. At various time points, combine the two in the microplate. After all points have been added, filter the reactions at the same time. The last wells to be mixed will be the shortest incubation times.

Dissociation, which can be measured more conveniently using the filtration format than SPA, is performed by adding an excess amount of unlabeled competitor after a receptor/radioligand mixture has reached steady state (plateau on the association curve). The figure below demonstrates an association/dissociation experiment (total binding only shown).

Results Analysis: Plot specific binding (total binding - nonspecific binding) versus time. Fit the association data to a one-phase exponential association curve and the dissociation data to a one-phase exponential decay curve. In the example above, a minimum reaction time of 2.5 hours would be adequate.

In addition to determination of the appropriate primary incubation time for steady state, a kinetic estimate for the equilibrium dissociation constant, Kd, can be made from the results of an association/dissociation experiment.

Association Experiment:

• Obtain kobs, expressed in min-1, from the nonlinear regression analysis of data

Dissociation Experiment:

• Obtain koff , expressed in min-1, from the nonlinear regression analysis of data

Calculate association rate constant, kon (in Molar-1 min-1)

Calculate equilibrium dissociation constant, Kd (in Molar):

Kd = koff/kon

Receptor Concentration - Zone A

Setup: Measure total binding (receptor + radioligand) and nonspecific binding (receptor + radioligand + excess unlabeled competitor) at various levels of added receptor.

Results Analysis: Plot total, NSB and specific binding (total - NSB) versus receptor amount. Plot total bound/total added expressed as a percent versus receptor concentration. Determine the level of receptor that yields <10% total binding/total added (Zone A).

See the Receptor Concentration - Zone A section in the SPA part of this document for further details and an example.

Solvent Tolerance

Setup: Measure total binding (receptor + radioligand) and nonspecific binding (receptor + radioligand + excess unlabeled competitor) at various concentrations of DMSO (or other solvent) using the determined optimum incubation time and optimum receptor concentration.

Results Analysis: Plot total and NSB versus final concentration of solvent

See the Solvent Tolerance section in the SPA part of this document for further details and an example.