Analysis Affinity of Cysteinyl Leukotrienes (CysLTs) by KinExA (CAT#: STEM-MB-0029-CJ)

Introduction

Cysteinyl leukotrienes (CysLTs) are a small family of biological signaling lipids produced by active leukocytes that contribute to diverse inflammatory disease states as a consequence of their engagement with dedicated G protein-coupled receptors. Immunization of mice with a CysLT-modified hapten carrier protein yielded novel monoclonal antibodies that display variable binding affinity to CysLTs. The cysteinyl leukotrienes (CysLTs) , leukotriene (LT) C4, LTD4, and LTE4, are biologically active lipids involved in both acute and chronic inflammatory responses that have been shown to contribute to inflammatory bowel disease and asthma. CysLTs were initially identified as the slow reacting substances of anaphylaxis.

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Principle Applications Procedure Materials Notes Related Products

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.

Materials

• Sample Type: Antibodies, Cells, Small Molecules, Proteins, DNA, Lipids, Viruses, Serum, & More
• Equipment: Kinetic Exclusion Assay (KinExA)

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