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a position-sensitive diode array detector (A). When antigen is injected through the flowcell, it interacts with the immobilized antibody, thus increasing the refractive index. Because of the very short distance the evanescent wave penetrates into the medium, only binding events very close to the sensorship surface are registered and the bulk of the protein solution contributes insignificantly to the signal (a 'bulk' effect can be seen at high concentrations and is constant throughout a protein injection). Upon binding, the angle of incident light at which SPR occurs changes (02) and this is again registered by the detector (B). The changes in angle (C) are converted by the instrument's software into arbitrary resonance units which are plotted versus time to provide a binding curve called a sensorgram (D). The sampling rate can be set by the user and can be as high as 10 points per second, providing a real-time response. Quantitative analysis of the binding data is performed by suitable software.

Figure 2 shows examples of antigen binding to a panel of different monoclonal antibodies which are immobilized on the sensorchip. Following injection of the antigen, buffer flow is introduced resulting in dissociation of bound material and a corresponding drop in the signal. This part of the sensorgram can be used to measure the dissociation rate constant of an interaction, whereas the 'on' phase can be analyzed for quantitation of the association rate constant. Figure 2 demonstrates that different antibodies

Figure 2 An example of antibody-antigen interaction measured by SPR. The same antigen was injected separately over three immobilized monoclonal antibodies. The arrows point to the beginning and the end of the antigen injection, which is followed by buffer flow. Note the differences between the antibodies in the association and dissociation rates. (Data kindly provided by Dr R. Karlsson, Biacore AB.)

Figure 2 An example of antibody-antigen interaction measured by SPR. The same antigen was injected separately over three immobilized monoclonal antibodies. The arrows point to the beginning and the end of the antigen injection, which is followed by buffer flow. Note the differences between the antibodies in the association and dissociation rates. (Data kindly provided by Dr R. Karlsson, Biacore AB.)

have distinct on and off rates for the same antigen. The equilibrium binding constant of an interaction can also be determined by injecting a series of concentrations and allowing the reaction to proceed to equilibrium. The response can then be plotted versus the concentration and suitable fitting algorithms can be used for quantitation.

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