Navigation Links
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

Kd 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 Kd value of 383 pM (95% confidence intervals 318 to 447 pM) was estimated directly from the curve and Bmax 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 Ki 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 Ki 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.

Zanalysis
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).



back to top
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. The development of a melanocortin MC4 receptor binding assay using the LEADseeker Multimodality Imaging System
2. The development of a melanocortin MC5 receptor binding assay using the LEADseeker Multimodality Imaging System
3. The development of a calcitonin-gene related peptide (CGRP) receptor-binding assay using the LEADseeker Multimodality Imaging System
4. The development of a somatostatin sst4 receptor-binding assay using the LEADseeker Multimodality Imaging System
5. The development of a serotonin 5-HT2C receptor-binding assay using the LEADseeker Multimodality Imaging System
6. Enzyme Immunoassay for Studying Intracellular Levels of cAMP
7. LiquiChip Kits for bead-based cytokine and kinase assays
8. Quality control of antibodies using the 2100 bioanalyzer and the Protein 200 Plus assay
9. High-content screening of GPCR activation with MetaXpress, AcuityXpress and the Transfluor assay system
10. Bio-Plex Cytokine Immunoassays and ELISA: Comparison of Two Methodologies in Testing Samples From Asthmatic and Healthy Children, Rev A
11. Principles of Curve Fitting for Multiplex Sandwich Immunoassays, Rev B
Post Your Comments:
(Date:12/24/2014)... CA (PRWEB) December 23, 2014 Pipette.com ... where their Twitter followers can submit their #HolidayInTheLab pictures ... and their lab mates. , For those struggling to ... mates, the Pipette.com Holiday in the Lab Contest provides ... Pipette.com Twitter followers to send pictures of their ...
(Date:12/24/2014)... Dec. 23, 2014 Sigma-Aldrich Corporation (NASDAQ: ... waiting period under the Hart-Scott-Rodino Antitrust Improvements Act ... on December 22, 2014, thereby completing the U.S. ... the acquisition of the Company by Merck KGaA, ... satisfies another condition to closing the transaction, which ...
(Date:12/22/2014)... ROCKVILLE, Md. , Dec. 22, 2014 ... MKT: SYN), a developer of pathogen-specific therapies ... focus on protecting the microbiome, today announced ... a Phase 1a clinical trial of SYN-004, ... the prevention of Clostridium difficile (C. ...
(Date:12/22/2014)... 2014  ( www.competitivehealth.com ) — Competitive Health is ... review and advocacy service, has signed an agreement to ... discount health services marketplace. 63% of Americans ... they expected to pay. As part of an ongoing ... Savings is pleased to offer medical bill review and ...
Breaking Biology Technology:Major Pipette Distributor Pipette.com Announces their Holiday in the Lab Contest 2Acquisition of Sigma-Aldrich by Merck KGaA, Darmstadt, Germany, Receives U.S. Antitrust Clearance 2Acquisition of Sigma-Aldrich by Merck KGaA, Darmstadt, Germany, Receives U.S. Antitrust Clearance 3Synthetic Biologics Announces Positive Topline Results from Phase 1a Trial of SYN-004 for the Prevention of C. difficile Infection 2Synthetic Biologics Announces Positive Topline Results from Phase 1a Trial of SYN-004 for the Prevention of C. difficile Infection 3Synthetic Biologics Announces Positive Topline Results from Phase 1a Trial of SYN-004 for the Prevention of C. difficile Infection 4Synthetic Biologics Announces Positive Topline Results from Phase 1a Trial of SYN-004 for the Prevention of C. difficile Infection 5WellCard Savings Announces CoPatient Medical Bill Review Service for Members 2
... June 6 Orexigen(R) Therapeutics, Inc. (Nasdaq: ... obesity, today announced positive secondary findings from the first of ... (naltrexone sustained release (SR)/bupropion SR). These data were presented ... Diabetes Association,s (ADA) 69th Scientific Sessions in New Orleans, Louisiana. ...
... and durability have always moved in opposite directions ... example, information carved in stone is not dense but ... chips can hold their information for only a few ... Berkeley National Laboratory (Berkeley Lab) and the University of ...
... Normal & Pathologic Biomolecules SANPB , Inserm / UEVE ... Centre of Mines-ParisTech (Evry, France), the NRG - UMR ... the Physic Institute of Stuttgart University (Germany) discovered a ... Results are published in Nanotechnology June10 2009 ...
Cached Biology Technology:Orexigen(R) Therapeutics Presents Additional NB-302 Contrave(R) Data at American Diabetes Association (ADA) 69th Scientific Sessions 2Orexigen(R) Therapeutics Presents Additional NB-302 Contrave(R) Data at American Diabetes Association (ADA) 69th Scientific Sessions 3Orexigen(R) Therapeutics Presents Additional NB-302 Contrave(R) Data at American Diabetes Association (ADA) 69th Scientific Sessions 4Orexigen(R) Therapeutics Presents Additional NB-302 Contrave(R) Data at American Diabetes Association (ADA) 69th Scientific Sessions 5A billion-year ultra-dense memory chip 2A billion-year ultra-dense memory chip 3A breakthrough toward industrial production of fluorescent nanodiamonds 2
(Date:12/10/2014)... Dec. 9, 2014  Valencell, a leader in performance ... a staggering demand from its licensees for highly accurate, ... is not solely coming from fitness and health sectors, ... "A wearable is only as useful as the ... the ultimate driver in long-term mass consumer adoption of ...
(Date:12/5/2014)... 2014 Increased emphasis on product quality, ... testing and inspection technologies. The convergence of industries ... range of innovative test solutions. This trend is ... which is generally more inclined towards communication, media ... strategies of test equipment vendors will cater to ...
(Date:11/21/2014)... -- According to a new market research ... Access Control, Intrusion Detection, Parking Management, Under Vehicle Inspection), ... Geography - Global Forecasts to 2020", published by MarketsandMarkets, ... around $25 Billion in 2014 and is expected to ... of 8.69%. Browse 116 market data Tables ...
Breaking Biology News(10 mins):Wearable Technology Products Demand Highly Accurate Biometric Technology 2Emerging Technologies Enable Smarter Monitoring of Assets, Push Industrial Testing, Inspection and Monitoring Market 2Emerging Technologies Enable Smarter Monitoring of Assets, Push Industrial Testing, Inspection and Monitoring Market 3Emerging Technologies Enable Smarter Monitoring of Assets, Push Industrial Testing, Inspection and Monitoring Market 4Industrial Security Systems Market Worth $38 Billion by 2020 2Industrial Security Systems Market Worth $38 Billion by 2020 3
... most of Costa Rica,s rainforests were cut down, researchers from ... thought was impossible - restoring a tropical rainforest ecosystem. ... with a sampling of local trees, native species began to ... can one day be replaced. Carl Leopold and his ...
... graduate student, the Forest Service, and California Department of ... captured this past week. A variety of hair, ... to determine if these were from a wolverine. After ... a remote camera on Feb. 28, 2008, in the ...
... PHILADELPHIA A new study by University of Pennsylvania ... humans, that ventilators combined with diaphragm disuse contributes to ... eighteen hours. Muscle atrophy in the diaphragm is a ... mechanical ventilation often have difficulty breathing after being removed ...
Cached Biology News:Can you rescue a rainforest? The answer may be yes 2Additional evidence of wolverine found in the Tahoe National Forest 2Additional evidence of wolverine found in the Tahoe National Forest 3Additional evidence of wolverine found in the Tahoe National Forest 4Long-term use of mechanical ventilation contributes to the deterioration of human diaphragm muscle 2
... through 63 ligand binding ... a broad therapeutic range. ... many non human tissue ... assays. Used to ...
...
... Sequencer 20 System revolutionizes DNA sequencing, delivering ... The Genome Sequencer 20 System includes: ... and consumables for library construction, amplification, and ... de novo assembly The ...
... to the maintenance of appropriate levels ... important and diverse as those involved ... stress response, antigen presentation, modulation of ... cell cycle regulation, transcription, and signalling ...
Biology Products: