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Section5:Assay Conditions
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Incubation Time - Signal Stability
Setup: Measure total binding (receptor + radioligand + SPA beads) and nonspecific binding (receptor + radioligand + excess unlabeled competitor + SPA beads) at various times using repetitive counting on the microplate scintillation counter.
Results Analysis: Plot total, NSB and specific binding (total binding - NSB) versus time
Since steady state will require a longer time to reach at lower concentrations of radioligand, these experiments are usually performed at radioligand concentrations below the Kd (i.e. 1/10 Kd) if signal strength permits. In addition, the total concentration of radioligand bound should be equal to less than 10% of the concentration added to avoid ligand depletion. The receptor concentration added must be lowered if this condition is not met.
This experiment is used to determine when a stable signal is achieved and how long a stable signal can be maintained. The signal is a combination of receptor/ligand reaching steady state and bead settling conditions. As SPA beads become packed at the bottom of the well, the efficiency of counting (particularly with 125I) increases. Therefore, it is important to determine when a uniform signal is obtained and adopt this time window as standard practice. In many assays. 8-16 hours are required for stable signal counting. Use approximately 0.125-0.5 mg SPA beads depending on results from preliminary experiments.
An example of an incubation time course is shown below. A minimum of 10 hours incubation time was chosen in this example and the interaction was stable for at least 24 hours. Failure to operate a receptor/ligand binding assay at steady state conditions may result in erroneous calculations for binding constants (Kd or Ki).
Receptor Concentration - Zone A
Setup: Measure total binding (receptor + radioligand + SPA beads) and nonspecific binding (receptor + radioligand + excess unlabeled competitor + SPA beads) at various levels of added receptor (typical μg amounts vary depending on the source and purity of 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).
It is ideal to keep the total amount of radioligand bound at less than 10% of the total amount added to avoid ligand depletion. This is considered the acceptable limit and is referred to as “Zone A”. Saturation experiments must be performed at <10% total ligand binding at all concentrations tested (0.1 x Kd to 10 x Kd), so an initial protein variation experiment at a radioligand concentration that is 0.1 x Kd is typically performed.
The example shown below uses radioligand at < 0.1 Kd and an increasing amount of membrane receptor protein. Two plots are shown: (Left) raw SPA data for total, NSB and specific; (Right) total bound/total added expressed as a percent. In this example, receptor levels less than 1.7 μg/well would meet Zone A requirements.
Total counts added (using liquid scintillation counting) are determined differently than the bound counts (SPA), therefore, in order to plot the % Total Bound/Added, the efficiency for each method must be taken into account, and any CPM data converted to DPM (described in APPENDIX). You cannot compare the CPM data from one instrument/scintillation method to that of another. The Section entitled “DPM Mode for SPA” demonstrates a representative method for determining efficiency for SPA bead counting. DPM for liquid scintillation counting can be obtained from the instrument directly. The stable signal count time must be determined prior to these experiments. If the signal dips after a high concentration of receptor, then the SPA beads may be in limited amounts.
SPA Bead Amount
Setup: Measure total binding (receptor + radioligand + SPA beads) nonspecific binding (receptor + radioligand + excess unlabeled competitor + SPA beads) and non-proximity effects (radioligand + SPA beads) at various SPA bead levels (typically 0.125 mg to 1.5 mg) using the determined optimum incubation time and optimum receptor concentration.
Results Analysis: Plot total, NSB, NPE, and specific binding (total - NSB) versus SPA bead amount. Choose a bead concentration beyond the linear range, at or near the initial saturation level on the specific binding curve.
Non-proximity effects (NPE) can be determined in the absence of added receptor. Ideally, the NPE signal would be identical to the nonspecific signal in the presence of unlabeled competitor. A level of SPA beads at 0.35 mg - 0.5 mg would provide the best economical signal for this example.
Solvent Interference
Setup: Measure total binding (receptor + radioligand + SPA beads) and nonspecific binding (receptor + radioligand + excess unlabeled competitor + SPA beads) at various concentrations of DMSO (or other solvent) using the determined optimum incubation time, optimum receptor concentration and optimum SPA bead amount.
Results Analysis: Plot total and NSB versus final assay concentration of DMSO
If the developed SPA receptor binding assay will be used to test organic compounds, interference with DMSO will need to be determined. As shown in the example data below, there can be significant signal reduction if the DMSO concentration becomes too high.
The level of DMSO in a SPA binding assay is determined by data in experiments such as the one in the example above and by the requirement set to maintain compound solubility.
Additional solvents (methanol, ethanol, etc.) or other agents (i.e. β-cyclohexadextrin) may need to be tested if compounds will be received in these other diluents.
Once determined, the solvent should be included in any further assay development or compound testing, including controls.
As an additional verification of minimal solvent interference, test competitive binding with a known competitor in the absence or presence of solvent at the determined level to be used in assays. Ideally, the test compound will have high affinity for the receptor and be freely soluble in aqueous buffer. The IC50 should not change in the absence or presence of the solvent.
