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Analysis Affinity of Anti-PD-1 by KinExA (CAT#: STEM-MB-0023-CJ)

Introduction

Programmed cell death protein 1 (PD-1) is currently the most widely exploited checkpoint blockade target. PD-1 is induced on T cells following activation and inhibits the signals of the T cell receptor and costimulatory molecules such as CD28,slowing an ongoing effector immune response to prevent immune-mediated damage. Checkpoint blockade immunotherapy of cancer seeks to prevent these inhibitory signals from slowing the immune response, reinvigorating effector T cell activity, and promoting tumor cell killing. Anti-PD-1 monoclonal antibodies (mAbs) have achieved widespread clinical success, but the relationship between binding affinity and functional efficacy of clinical anti-PD-1 antibodies is unpredictable.




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 Proteins, DNA, Viruses, Serum, & More
• Equipment: Kinetic Exclusion Assay (KinExA)
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