title: Measuring Sphingosine-1-Phosphate/Protein Interactions with the Kinetic Exclusion Assay.
abstract: By directly detecting the ligand-free binding sites in a sample, the kinetic exclusion assay (KinExA®) provides a compelling alternative to SPR-based techniques for determining equilibrium dissociation constants of protein-ligand interactions. It is especially useful for observing protein-lipid interactions, as binding of native lipids occurs entirely in solution, and monoclonal antibodies can be used to directly compete with a protein of interest for lipid binding. By measuring the antigen-free binding sites on the antibody and using competition affinity analysis, the Kdfor the lipid binding the protein and the antibody can be determined simultaneously. Herein, we describe this label-free approach for determining the Kdfor S1P-binding serum albumin, which chaperones ~30% of the S1P in human plasma.
authors.affiliation: Lpath Incorporated, 4025 Sorrento Valley Blvd, San Diego, CA, 92121, USA.
authors.forename: Jonathan K
authors.initials: JK
authors.lastname: Fleming
authors.affiliation: Lpath Incorporated, 4025 Sorrento Valley Blvd, San Diego, CA, 92121, USA. jonwojciak@gmail.com.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Wojciak
mesh_terms: D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D008246:Lysophospholipids / Q000378:metabolism*; D011485:Protein Binding; D000075462:Serum Albumin, Human / Q000737:chemistry / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D008246:Lysophospholipids; C060506:sphingosine 1-phosphate; D013110:Sphingosine; D000075462:Serum Albumin, Human
keywords: Anti-lipid antibody; Bovine serum albumin; Competitive affinity analysis; Human serum albumin; Kinetic exclusion assay; Physical biochemistry; Sphingosine-1-phosphate