Analysis Affinity of Clinical Anti-drug Antibody (ADA) by KinExA (CAT#: STEM-MB-0021-CJ)

Introduction

The affinity dissociation constant (KD) of ADA is an important attribute which can represent the kinetics of antibody development and has rarely been included in immunogenicity assessments of biotherapeutics. European Medicines Agency (EMA) guidance states affinity characterization of ADA response may be required on a case-by-case basis, and assays used for these measurements should be qualified for their intended purpose. Challenges to affinity determination in clinical ADA samples include a combination of assay sensitivity due to the relatively low concentrations of ADA, matrix interference from serum proteins, residual drug, and the polyclonal nature of the ADA response.

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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: Serum, Plasma, Urine
• Equipment: Kinetic Exclusion Assay (KinExA)

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