Analysis Affinity of Jedi067 to IL-1RI by KinExA (CAT#: STEM-MB-0014-CJ)

Introduction

Antibodies are routinely engineered for higher affinity using a variety of different molecular techniques that typically incorporate mutagenesis leading to changes in the primary amino acid sequence, alongside methodologies for selecting the improved variant. In the case of antibody affinity maturation, examples of affinity gains of greater than >10,000-fold and final dissociation constants (KD) in the low picomolar range have been described using different display technologies. IL-1RI is an 80 kDa membrane-associated protein and a member of the IL-1R/Toll-like receptor (TLR) superfamily. IL-1RI has three ligands, interleukin-1alpha (IL-1α) , interleukin-1beta (IL-1β) , and an inhibitory ligand, interleukin-1 receptor antagonist (IL-1Ra). And IL-1RI is also a potential target for therapeutic blockade.

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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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