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

Journal of Leukocyte Biology

Beatriz Vega⁴  Laura Martínez Muñoz⁴  Borja L. Holgado⁴  J. M. Rodríguez-Frade⁴  Mario Mellado⁴  José F. Rodríguez–⁵  Ana Calle⁶  J. L. Rodríguez-FernÃndez³  Pilar Lucas⁴  Laura M. Lechuga¹  Ricardo Gutiérrez-Gallego and² 

[1] de Investigación en Nanociencia y Nanotecnologia (CIN)/Consejo Superior de Investigaciones Cientificas, Campus UAB Bellaterra, Barcelona, Spain;Bioanalysis Group, Neuropsychopharmacology Program, IMIM-Hospital del Mar and Department of Experimental and Health Sciences, University Pompeu Fabra (UPF), Barcelona Biomedical Research Park (PRBB), Barcelona, Spain Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Cientificas, Campus de Cantoblanco, Madrid, Spain;Centros de Investigaciones Biológicas and Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Cientificas, Campus de Cantoblanco, Madrid, Spain;Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Cientificas, Campus de Cantoblanco, Madrid, Spain;–CIBER-BBN, Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Cientificas, Barcelona, Spain; IMM-Instituto de Microelectrónica de Madrid/Consejo Superior de Investigaciones Cientificas, Madrid, Spain;

关键词: chemokine;    biosensor;    glycosaminoglycan;    affinity constant;    chemokine receptor;   

DOI  :  10.1189/jlb.1010565

学科分类：生理学

来源: Federation of American Societies for Experimental Biology

【 摘 要 】

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.

【 授权许可】

Unknown