( Key words: ...)The development of a CXCR2 chemokine receptorbinding assay using the LEADseeker Multimodality Imaging System

Key words: CXCR2 • GPCR • receptor binding assay • LEADseeker • SPA Imaging Beads

Chemo-attractant cytokines (chemokines) are low molecular weight, pro-inflammatory cytokines with potent leukocyte activation and/or chemotactic activity in vivo (1). Chemokines are classified into four families (CXC, CC, C, and CXC3) based on the number and sequential relationship of the first two of four conserved cysteine residues. They induce their biological effect by binding to chemokine receptors that belong to the superfamily of G protein-coupled seven-transmembrane domain receptors (2).

The CXC chemokines are pivotal mediators that activate or inhibit angiogenesis by interacting with cognate receptors expressed by endothelial cells (3). CXCR2 is a major chemokine receptor expressed in neutrophils and binds chemokines with a Glu-Leu-Arg (ELR) sequence in the amino terminus; IL-8, GRO-α, GRO-β, GRO-γ, NAP-2, ENA-78, and GCP-2 (4).

This application note describes a 384-well CXCR2 chemokine receptor-binding assay which has been developed using the LEADseeker™ Multimodality Imaging System.

Materials
Products used
LEADseeker Multimodality Imaging System 18-1140-71

Wheat Germ Agglutinin (WGA) PS SPA RPNQ0260
Imaging Beads

[¹²⁵I]GRO-a IM305

Other materials required
Human recombinant CXCR2 receptor membrane preparation (Euroscreen ES-145-M)

Human recombinant IL-8 (Sigma 123K0714)

Human recombinant GRO-α(Sigma G0657)

Costar™ solid white 384-well NBS™ coated assay plate (Corning 3652)

EDTA-free protease inhibitor cocktail tablets (Roche 1873580)

Buffer: 25-mM HEPES pH 7.4, 5-mM MgCl₂, 1-mM CaCl₂, 0.2% (w/v) BSA, protease inhibitors (one tablet per 50-ml buffer).
Note: that BSA and protease inhibitors were added freshly on the day of use.

GraphPad Prism™ software (GraphPad Software)

Protocol
1. Human recombinant CXCR2 receptor membrane preparation was used in conjunction with [¹²⁵I]GRO-αligand and WGA PS SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 125-nM IL-8. The standard assay format was as follows

2. Reagents were added in the following order: Buffer or DMSO solution, unlabeled ligand (NSB wells), labeled ligand, premixed bead, and membrane. Total assay volume was 40 µl

3. Diluted membrane and bead were premixed at room temperature immediately prior to assay addition.

4. Wells contained 10 µl of 1.48-nM [¹²⁵I]GRO-α(final concentration 0.37 nM) unless otherwise stated. Wells also contained 0.1 mg of beads and 1.2 µg of membrane, which were added together in a 20-µl volume.

5. NSB wells contained 10 µl of 0.5-µM IL-8 (final concentration 0.125 µM) in addition to the above.

6. For DMSO tolerance studies, DMSO was diluted in binding buffer to give final concentrations per well as shown in the results.

7. Plates were sealed and incubated in darkness for 8 h at room temperature (20–25 ºC) unless otherwise stated.

8. Following incubation, plates were imaged on the LEADseeker Multimodality Imaging System for 3 min with quasi-coincident averaging and 3 ×3 binning.


Results
K_d determination
Saturation binding was performed with dilutions of [¹²⁵I]GRO-αto give a range of concentrations from 2.5 to 2000 pM in the wells. Figure 1 shows the saturation curve that was fitted using non-linear regression with the data analysis software package GraphPad Prism v4.0. A K_d value of 383 pM (95% confidence intervals 318 to 447 pM) was estimated directly from the curve and B_max was determined at 6 pmol/mg of protein.

DMSO tolerance
Dilutions of DMSO were prepared in assay buffer to give a range of concentrations from 2% (v/v) to 25% (v/v) in the wells. From the results shown in figure 2 it can be seen that the assay was tolerant to DMSO up to a final concentration of 12.5% (v/v)

IC₅₀ determination
Competitive binding of 0.37-nM [¹²⁵I]GRO-αwith unlabeled IL-8 and GRO-αwas assessed and the IC₅₀ values calculated as shown in Figure 3. DMSO was included at an assay concentration of 2.5% (v/v). Final concentrations in the well for unlabeled GRO-αwere 0.03 to 440 nM, and for unlabeled IL-8, 0.03 to 500 nM.

The IC₅₀ value for GRO-αwas determined to be 2 nM (95% confidence interval range 1.7 to 2.3 nM) and the K_i value was 1.02 nM (95% confidence intervals 0.87 to 1.19 nM). The IC₅₀ value for IL-8 was 11 nM (95% confidence interval range 10 to 12 nM) and the K_i was 5.68 nM (95% confidence intervals 5.14 to 6.27 nM).

Time course
A time course was performed using standard reagent concentrations as detailed in the protocol and DMSO included at an assay concentration of 2.5% (v/v). Readings were taken over an increased incubation time of 18 h. The assay reached equilibrium after approximately 7 h and was stable for at least a further 5 h as demonstrated by the results shown in Figure 4.

Z′analysis
Finally, a Z′analysis was performed using 48 replicate values each for "total" and NSB wells, Z′was determined to be 0.83 (Fig 5), which confirmed the robustness of the assay (5).

Conclusions
The CXCR2 chemokine receptor-binding assay has been successfully miniaturized to a 384-well format using the LEADseeker Multimodality Imaging System and is suitable for adaptation to automated screening formats. The assay is tolerant to DMSO up to a concentration of 12.5% (v/v), robust; achieving a Z′value of 0.83, and has a stability window of at least 5 h.

References
1. Rossi, D. and Zlotnik, A. Annual Review Immunology 18, 217–242 (2000).

2. Premack, B. A. and Schall, T. J. Nature Medicine 2, 1174–1178 (1996).

3. Addison, C. L. et al. Journal of Immunology, 165, 5269–5277 (2000).

4. Clark-Lewis, I. et al. Journal Biological Chemistry 278 (1), 289–295 (2003).

5. Zhang, J. et al. Journal Biomolecular Screening 4 (2), 67–73 (1999).

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