Analysis Affinity of Denosumab and Human OPG-Fc/RANKL Fragment by KinExA (CAT#: STEM-MB-0044-CJ)

Introduction

RANKL is an important mediator for osteoclast formation, function and survival. The catabolic action of RANKL is inhibited by osteoprotectin (OPG) , a soluble decoy receptor that binds to RANKL and thus prevents the activation of its receptor RANK, which is present on osteoclasts and osteoclast precursors. Human OPG structures have been valuable research tools because they effectively inhibit bone resorption in various species. Dinosemer (AMG 162) is a fully human monoclonal antibody that binds to and inhibits RANKL. Dinosemet reduced biochemical markers of bone resorption in postmenopausal women with low bone mass and in patients with multiple myeloma or bone metastases.

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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

Pharmacokinetics & pharmacodynamics.

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

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