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Analysis Affinity of Fabs (Mouse and Human) for Dabigatran by KinExA (CAT#: STEM-MB-0030-CJ)

Introduction

Thromboembolic disorders such as myocardial infarction, stroke, and venous thromboembolism are the most common causes of mortality and morbidity in Western societies.The dabigatran etexilate, is a novel, potent, nonpeptidic direct thrombin inhibitor. The orally administered double prodrug is hydrolized in vivo by esterases into the active form, dabigatran. It can bind reversibly to the active site of both free and clot-bound thrombin, thus effectively preventing the formation of fibrin clots for the prevention of stroke in patients with atrial fibrillation.

Antibodies and antibody fragments such as fragment antigen binding (Fab) can be used as antidotes for toxins and drugs. With Fabs, intoxications with digoxin and colchicine can be treated and the effect of desipramine can be reversed.




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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