Analysis Affinity of Human Monoclonal Antibody (MAb) 2G12 to Envelope Glycoprotein (gp120) by KinExA (CAT#: STEM-MB-0045-CJ)

Introduction

Human monoclonal antibody (MAb) 2G12 efficiently neutralizes a broad range of HIV-1 primary isolates in vitro and protects from in vivo viral challenge in macaques in combination with other antibodies. MAb 2G12 binds with high affinity to a unique, conserved epitope on the HIV-1 envelope that is formed by a cluster of N-linked, high-mannose glycan groups on the “silent” face of gp120. Since the identification of its epitope, MAb 2G12 has received significant attention as a template for HIV-1 vaccine development.

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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; Virology

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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