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Analysis Affinity of ICOSL and BAFF with AMG 570 by KinExA (CAT#: STEM-MB-0040-CJ)
Introduction
Autoimmune diseases, including systemic lupus erythematosus (SLE) , are associated with dysregulated T and B cell responses. AMG 570 is a bi-specific molecule that targets T cell and B cell activity by inhibiting inducible costimulatory ligand (ICOSL) and B cell activator (BAFF). Dual inhibition of ICOSL and BAFF was more effective than single inhibition in improving the incidence and severity of arthritis in the mouse CIA model as well as reducing anti-DSDNA IgG, delayed albuminuria, and improving survival in the NZB/NZW lupus model. In addition, a single dose of AMG 570 was safe and well tolerated, and showed pharmacodynamic activity consistent with the inhibition of ICOSL and BAFF.
Principle
KinExA is a two-stage analysis system. In the first stage, a number of solutions are prepared, where one partner remains constant (constant binding partner, or CBP) and the other (titrant) is variable, usually serially diluted. As the titrant is added, the free CBP decreases and is analysed by a sophisticated and precise microfluorescence measurement device. The signal generated can be mathematically related to the affinity (KD) of the two molecules for each other, as well as the kinetic parameters of binding (kon) and dissociation (koff).
Applications
Immunology/Inflammation, Toxicology; Pharmacology
Procedure
1. preparation of the functionalized beads which will capture the analyte for measuremen.
2. preparation of a series of solutions consisting of a constant initial concentration of one component of the binary reaction and serial dilutions of the other reactant. The component that is kept constant is the constant binding partner (CBP) , and is the one which will be analyzed.
3. each reaction mixture is sampled and the fluorescence of free CBP bound to the capture beads is obtained for subsequent numerical analysis.
2. preparation of a series of solutions consisting of a constant initial concentration of one component of the binary reaction and serial dilutions of the other reactant. The component that is kept constant is the constant binding partner (CBP) , and is the one which will be analyzed.
3. each reaction mixture is sampled and the fluorescence of free CBP bound to the capture beads is obtained for subsequent numerical analysis.
Materials
• Sample Type: Antibodies, Cells, Small Molecules, Lipids, Serum (Human and Mouse)
• Equipment: Kinetic Exclusion Assay (KinExA)
• Equipment: Kinetic Exclusion Assay (KinExA)
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