Analysis Affinity of Antibody-RTX751 by KinExA (CAT#: STEM-MB-0050-CJ)

Introduction

The adenylate cyclase toxin (ACT) is frequently cited as a leading candidate for inclusion in next generation vaccines,but is not included in any current acellular vaccines due to it being unavailable at the time those vaccines were being developed and to poor understanding of its role in protection. In addition, ACT is a large, multi-domain protein that targets phagocytic leukocytes expressing the αMβ2 integrin (also known as CR3, Mac-1 and CD11b/CD18) through its Cterminal repeat-in-toxin (RTX) domain. Protect bacteria from innate immune responses during the early stages of infection by inhibiting bacterial phagocytosis.The C-terminal RTX domain can recapitulate many of the responses resulting from immunization with ACT, while possessing superior biochemical characteristics.

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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

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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