Technical Advance: Surface plasmon resonance-based analysis of CXCL12 binding using immobilized lentiviral particles

• Published in: Journal of leukocyte biology Vol. 90; no. 2; pp. 399 - 408
• Main Authors: Vega, Beatriz, Muñoz, Laura Martínez, Holgado, Borja L, Lucas, Pilar, Rodríguez-Frade, J M, Calle, Ana, Rodríguez-Fernández, J L, Lechuga, Laura M, Rodríguez, José F, Gutiérrez-Gallego, Ricardo, Mellado, Mario
• Format: Journal Article
• Language: English
• Published: United States Society for Leukocyte Biology 01.08.2011
• Subjects: affinity constant; Biosensing Techniques - methods; biosensor; chemokine; Chemokine CXCL12 - analysis; Chemokine CXCL12 - metabolism; chemokine receptor; glycosaminoglycan; Glycosaminoglycans - pharmacology; Heparin - pharmacology; Humans; Immobilized Proteins; Kinetics; Lentivirus - metabolism; Protein Binding; Receptors, CXCR4 - metabolism; Surface Plasmon Resonance
• ISSN: 0741-5400; 1938-3673
• DOI: 10.1189/jlb.1010565

New method to determine chemokine binding parameters using the native conformation of the receptor and a surface plasmon resonance‐based strategy. Use of SPR‐based biosensors is an established method for measuring molecular interactions. Their application to the study of GPCRs is nonetheless limited to detergent‐solubilized receptors that can then be reconstituted into a lipid environment. Using the chemokine receptor CXCR4 and its specific ligand CXCL12, we outline here a highly reproducible biosensor method based on receptor presentation on the surface of lentiviral particles; the approach is simple and does not require the use of antibodies to achieve correct receptor orientation on the sensorchip surface. We measured the kinetic parameters of CXCR4/CXCL12 binding in a single step and in real time and evaluated the effect of GAG presentation of chemokines on this interaction. The data indicate that at low concentrations, soluble heparin modulates CXCR4/CXCL12 interaction and at high concentrations, abrogates binding. These observations suggest that in addition to their known role in modulating local chemokine availability, GAG affect the receptor/ligand interaction, although their influence on affinity parameters is very limited. The method will also be useful for quantifying these biomarkers in biological fluids and for the development of high‐throughput screening for their antagonists.