Methods for affinity measurement

As already pointed out, affinity is measured when the equilibrium between the two molecules has been achieved in solution. Hence, measuring KA for monoclonal antibody and its antigen consists in mixing the monoclonal antibody (mAb) and the antigen at various initial concentrations, letting the equilibrium be established, measuring the concentrations of free and saturated sites at equilibrium and analyzing the binding curve. The experimental difficulty resides in distinguishing the free and bound state of either the monoclonal antibody or the antigen. Several methods can be used, such as equilibrium dialysis, radioimmunoassay (RIA) using precipitation with salts and other agents, filtration, fluorescence measurements and RIA- or ELISA-based methods. Equilibrium dialysis is the method of choice for hapten and dialyzable antigen but is not applicable for macromolecular antigens. Radioimmunoassays require radiolabeling of the antigen which may alter the binding properties of the antigen. Precipitation or filtration methods need the complex to be stable enough during the time required to achieve its separation so that the equilibrium is not disrupted. The use of fluorescence to determine KA requires that either the monoclonal antibody or the antigen be fluorescent, and that a change in fluorescence should occur upon formation of the monoclonal antibody/ antigen complex. The fluorescence signal used can be either intrinsic (e.g. tryptophan residues from the antibody and, possibly, from the protein antigens; some prosthetic groups such as pyridoxal-phosphate, NADH, NADPH, flavins, etc.) or result from prior fluorescent labeling of the antigen or the antibody with a fluorochrome. The fluorescent change observed upon association may be one of the following: excitation or emission wavelength shift, fluorescence quenching, fluorescence transfer, change in fluorescence polarization. As already underlined for radiochemical labeling, adding an extrinsic fluorescent label may affect the binding characteristics of the antigen or the antibody, either by steric hindrance or through a change in conformation, thus modifying the KA. The sensitivity of current fluori-meters sets a higher limit of 108-109 M"1 on the KA that can be determined. Some mAbs have much higher affinities for their Ag. Several methods such as indirect ELISA or surface plasmon resonance using immobilized antigen or monoclonal antibody have been developed to determine values of the affinity.

Such measurements yield real values of KA only rarely. Friguet and colleagues have defined a procedure to measure by a competition ELISA method the real association equilibrium constant in solution. This method requires no labeling of either the antigen or the antibody, which ensures that the affinity is characteristic of native antibody and antigen. This method gives access to affinity constants as high as 10,o-10"m~' and can be used for studies on protein-protein interactions that do not involve antibodies. Since this method has been frequently used for affinity determination, its principle is described in the next paragraph.

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