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Analysis Affinity of Antibodies to hIL-13Rα1 by KinExA (CAT#: STEM-MB-0008-CJ)

Introduction

Asthma is a heterogeneous and chronic inflammatory disease of the airways that is characterized by airway inflammation, reversible airway obstruction, mucus hypersecretion, airway hyper-responsiveness and airway wall remodelling.In a significant proportion of patients, the disease manifests as an allergic hypersensitivity dominated by an immune response polarized to the TH2 phenotype. This activation results in the secretion of TH2 cytokines [IL (interleukin) -4, IL-13 and IL-5] that either directly or indirectly trigger many of the pathologies associated with this disease including IgE production, eosinophilia, mucus secretion by lung epithelial cells and airway smooth muscle hyper-responsiveness.<br /><br />IL-13 can bind to and activate a receptor complexmade up of IL-13Rα1 (IL-13 receptor α1) and IL-4Rα (IL-4 receptor α). Several lines of evidence suggest that IL-13 and IL-13Rα1 are critically involved in asthmatic responses. Genetic studies in mice have shown that IL-13 plays critical roles in the regulation of IgE synthesis, mucus hypersecretion, sub-epithelial fibrosis and eosinophil infiltration into the lung, and it has been associated with the regulation of chemokine receptors including CCR5 (CC chemokine receptor 5).




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;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.

Materials

• Sample Type: Antibodies (Human and Mouse)
• Equipment: Kinetic Exclusion Assay (KinExA)