Section5:SPA Assay Format

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Plastic SPA beads, made of polyvinyltoluene (PVT), act as a solid solvent for diphenylanthracine (DPA) scintillant incorporated into the bead
A Glass SPA bead, or Yttrium silicate (YSi), uses cerium ions within a crystal lattice for the scintillation process. In general, YSi is a more efficient scintillator than PVT is, but YSi SPA beads are requires continuous mixing even during dispensing.

Typical experiments to investigate nonspecific binding of radioligand to SPA beads include varying the amount of radioligand (above and below the predicated Kd value) and the amount of SPA beads (0.1 mg to 1 mg) in the absence of added membrane protein. Results from this experiment can identify the proper type of SPA beads to use in future experiments, as well as the baseline background due to non-proximity effects. An example experiment using a kit from GE Healthcare (formerly Amersham Biosciences) that contains several different SPA bead types (Select-a-Bead kit, #RPNQ0250) is shown below.

For this example, which was performed in the absence of added membrane receptor, the PVT-PEI WGA Type A SPA beads yields the lowest interaction with the radioligand and was used for further assay development. An increase in signal with an increasing amount of added SPA beads is normal. Additives may be useful in decreasing high levels of nonspecific binding of radioligand to the SPA beads (see table of Agents which Reduce NSB in the Assay Buffer section).

Plate Type

The type of plate that is used for SPA receptor binding assays may be influenced by the following factors:

Counting instrument used (Trilux, TopCount, CLIPR, LeadSeeker)
Miniaturization (96-well, 384-well)
Binding of radioligand to plastics
Liquid dispensing/automation equipment

The table below lists typical choices for SPA assays:

Plate Type	Instrument	of Wells	Comments
Costar #3632	Trilux	96	White/Clear-bottom, 96-well
Costar #3604	Trilux	96	White/Clear-bottom, 96-well, non-binding surface (NBS), may be useful when ligands are sticky
PE LAS 401	Trilux	96	Clear/flexible, not amenable to automation or liquid dispensing instrumentation
Costar #3706	Trilux	384	White/Clear-bottom, 384-well
PE LAS Optiplate	TopCount	96	White/solid bottom, 96-well

The data shown below demonstrates an advantage of the NBS plates when using a radioligand, which binds nonspecifically to plate plastic.

69,000 CPM of 125I-labeled ligand added to the well, incubated for 60 min. Radioactivity removed and wells washed. SPA beads then added. Data demonstrates that a radioligand sticking to the plate surface can elicit an SPA signal. NBS plate yields significantly less nonspecific binding of radioligand.

Order of Addition

The order of addition for reagents may affect assay performance as well as ease of automation. Three basic formats have been used:

Method		Advantage
Membrane precoupled to SPA bead Time zero (T=0) addition of SPA beads Delayed addition of SPA beads		May aid in lowering NSB Easily automated Optimum ligand/receptor interaction possible

Time zero or delayed additions are the most commonly used formats in HTS, with time zero addition requiring fewer manipulation steps. Experiments may be required to determine the optimum method to be used for a particular receptor to maximize signal to background levels.

In addition, the effect of DMSO on intermediate reactants should be investigated. If compounds in DMSO are added into the wells first (most common method for screening efforts), other reagents added (i.e. radioligand, membranes, beads, etc.) may be affected by the concentration of DMSO, or if the time before reaching the final reaction mixture becomes significant.

Non-Specific Binding (NSB)/Non-proximity Effects (NPE)

In order to obtain the maximum signal to noise ratio possible for SPA receptor binding assays, it is important to understand the two different types of signals associated with the radioligand and SPA beads, which may contribute to the total assay background levels.

Non-Specific Binding (NSB) to SPA Beads

This signal is attributed to radiolabel which may adhere to the SPA beads themselves and not through a specific interaction with the receptor attached to the SPA bead (Left panel, below). This component of background signal can be determined in the presence of an excess concentration of competitor in the absence of the membrane receptor. Reduction of this factor can be accomplished through the careful use of buffering systems and the appropriate bead type. Determination of NSB to the SPA beads is separate from the NSB associated with membrane receptor preparations.

A competition experiment using an unlabeled compound in the absence or presence of added receptor may assist in identifying nonspecific binding problems.

Non-Proximity Effects (NPE)

NPE occurs when either the concentration of the radioligand or the concentration of SPA beads is sufficiently high enough to elicit a signal from the emitted β-particles. This can occur even though the labeled ligand is not attached directly to the SPA bead through the interaction with the receptor or the nonspecific interaction with the bead (Right panel, below). In general, this signal is a linear function, directly proportional to the concentrations of each of these reagents. Therefore, a careful balance between radiolabel and SPA beads is crucial to maximize signal and sensitivity while minimizing NPE and ultimately cost. The only technique available to minimize NPE is adjustment of the SPA bead or radiolabel concentrations.

For routine SPA binding assays, nonspecific binding may be a combination of nonspecific binding to SPA beads as well as nonspecific binding to the receptor, and are expressed as one. Total nonspecific binding is measured in the presence of an excess concentration of unlabeled competitor.

Temperature

Typically, receptor binding assays used in screening efforts are performed at room temperature. Comparison experiments may be required if other temperatures are considered. A kinetic analysis may be necessary as well. The data below depicts an SPA receptor binding assay performed at three temperatures.

Note: Since in nearly all cases, the microplate scintillation counter is at room temperature, and a 96-well plate requires approximately 16 minutes to read, it is difficult to perform SPA assays at temperatures other than room temperature. Data shown at the left was generated by incubation of a limited number of wells (n=4, different plates) at the indicated temperatures and counting them rapidly in the instrument. The information is useful in areas where there are significant variations in day-to-day laboratory temperatures.