SPA technology is widely used for receptor binding assays. The following summarizes data from receptor binding SPA developed for four newly released iodinated ligands: macrophage inflammatory protein-1α (MIP-1α), vasopressin, rat atrial natriuretic peptide and human α atrial natriuretic peptide.
Full Scatchard analysis was carried out, in order to determine a range of Kd values for the ligand / receptor combinations. These data were generated using particular cell lines under specific assay conditions. Customers should carry out their own validation using the cell line and system of their choice. Saturation curves were set up using a fixed receptor and bead concentrations with total binding and non specific binding (NSB) tubes (NSB data generated using excess cold ligand).
Scatchard analysis of the data was then used to generate Kd values. See figures for individual assay conditions. The Kd values for the four assays are shown in Table 1 and represent the ranges from the testing of several different batches of each ligand:
Table 1 : Kd values
Figure 1 Scatchard plot for [125I]Macrophage inflammatory protein-1α, IM285.
Increasing concentrations of [125I]MIP1α were incubated with 7μg human recombinant CCR1 chemokine receptor (Biosignal, cat no.. MCCRIH) and 1mg PVT-WGA SPA beads in a total assay volume of 170μl containing 50mM HEPES, 5mM MgCl2, 1mM CaCl2, 0.2 (w/v)% BSA, pH 7.4. NSB was determined in the presence of 76nM MIP-1α. Assays were performed in Beckman Biovials™ and incubated at room temperature for 60 minutes then overnight at 2-4°C prior to counting.
Figure 2 Scatchard plot for (3-[125I]-iodotyrosyl) vasopressin [Arg8], IM182.
Increasing concentrations of [125I] vasopressin were incubated with 37.4μg human recombinant vasopressin receptor type 1A (Biosignal, cat no. BSR MVIAH) and 1mg PVT-WGA SPA beads in a total assay volume of 250μl containing 50mM Tris.HCl, 5mM MgCl2, 0.01%(w/v) BSA, pH 7.4. NSB was determined in the presence of 1.3μM vasopressin (Bachem). Assays were performed in Beckman Biovials and incubated at room temperature for 60 minutes then overnight at 2-4°C prior to counting.
Figure 3 Scatchard plot for (3-[125I]iodotyrosyl28) atrial natriuretic peptide (rat), IM186.
Increasing concentrations of [125I] atrial natriuretic peptide were incubated with 50μg bovine adrenal cortex and 1mg PVT-WGA SPA beads in a total assay volume of 200μl containing 0.01M phosphate buffer, 2.7mM KCl, 0.137M NaCl, 0.1%(w/v) BSA, 5mM EDTA, pH 7.4. NSB was determined in the presence of 4.2μM atrial natriuretic peptide (Bachem). Assays were performed in Beckman Biovials and incubated at room temperature for 60 minutes then overnight at 2-4°C prior to counting.
Figure 4 : Scatchard plot for (3-[125I]iodotyrosyl28) atrial natriuretic peptide(human), IM187
Increasing concentrations of [125I] atrial natriuretic peptide were incubated with 25μg bovine adrenal cortex and 1mg PVT-WGA SPA beads in a total assay volume of 200μl containing 0.01M phosphate buffer, 2.7mM KCl, 0.137M NaCl, 0.1%(w/v) BSA, 5mM EDTA, pH 7.4. Non-specific binding was determined in the presence of 1.96μM atrial natriuretic peptide (Bachem). Assays were performed in Beckman Biovials and incubated at room temperature for 60 minutes then overnight at 2-4°C prior to counting.
Conclusion
The data shows further applications of SPA to receptor binding assays and the successful use of SPA in determination of kinetic data.