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Kinetic analysis of antibody–antigen interaction by Dual polarization interferometry (DPI) (CAT#: STEM-MB-0446-WXH)

Introduction

Characterisation of antibody affinity and kinetics is important for the selection of appropriate reagents for immunoassay. For equilibrium measurements antibody affinity determines the detection limit and for a number of antibodies with the same affinity for the antigen (Ag) the association rate constant will determine the speed of the assay. More fundamentally, changes in reaction rates and affinity can be used to probe function or structure in one of the reactants. This is of value when antibodies or antigens are modified, in recombinant protein characterisation and in the development of purification schemes.




Principle

Dual polarization interferometry (DPI) is an analytical technique that allows the simultaneous determination of thickness, density, and mass of a biological layer on a sensing waveguide surface in real time. DPI focuses laser light into two waveguides. One of these functions as the "sensing" waveguide having an exposed surface while the second one functions to maintain a reference beam. A two-dimensional interference pattern is formed in the far field by combining the light passing through the two waveguides. The DPI technique rotates the polarization of the laser, to alternately excite two polarization modes of the waveguides. Measurement of the interferogram for both polarizations allows both the refractive index and the thickness of the adsorbed layer to be calculated. These measurements can be used to infer conformational information about the molecular interactions taking place, as the molecule size (from the layer thickness) and the fold density (from the RI) change.

Applications

Kinetic analysis of antibody–antigen interaction.

Procedure

1. Setting of dual polarization interferometry
2. Preparing the DPI sensor chip
3. Immobilization of target on DPI biosensor
4. Reagent was injected to react
5. Quantitative analysis

Materials

• DPI biosensor
• DPI sensor chip
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