Affinity measurement in solution by competition ELISA

In the competition ELISA the monoclonal antibody at a fixed concentration and the antigen at varying concentrations are first incubated in solution until the equilibrium is reached. Then, the concentration of the free (i.e. not associated with antigen) monoclonal antibody at equilibrium is determined by a classical indirect ELISA using antigen-coated plates.

For correct determination of the free antibody concentration at equilibrium, several requirements must be fulfilled: 1) The absorbance obtained in the last step of the indirect ELISA, which reflects the free antibody concentration, must be proportional to the antibody concentration tested. 2) Only a small percentage of the free antibody molecules (i.e. less than 10%) must bind to the coated antigen, to prevent any significant disruption of the equilibrium in the liquid phase. This ensures that, during the ELISA, the equilibrium in solution is not significantly modified. By this means, the observed equilibrium constant corresponds to the real affinity. 3) Since the association constant KA is generally dependent on the temperature, the temperature must be kept constant throughout.

To satisfy requirements (1) and (2), it is necessary in previous ELISA experiments to determine the total (i.e. initial) antibody concentration range that must be used, the concentration of the coated antigen and the optimal incubation time of the antibody solutions in the coated wells. The state (native or partially denatured upon coating) of the coated antigen and whether or not it is recognized by the mAb differently from the soluble antigen is not important as long as the coated antigen can specifically and quantitatively trap the free antibody.

An example of affinity determination by the competition ELISA method described above is given in Figure 2 using a computerized fitting program.

The multivalency of antibodies and antigens

Figure 2 Saturation curve in solution of a monoclonal antibody by its antigen: [x]/[AbJ = [Ag]/([Ag] + Ka). The value of KD (1.5 x 10-9 M) was directly extracted with a computerized nonlinear regression method.

sometimes complicates the determination of affinity. The distortion of the saturation curves (as compared with simple binding) caused by the multivalence of immunoglobulins in indirect competition ELISA was first analyzed by Stevens and has become the object of much attention. However, the simple data analysis initially proposed by Friguet and colleagues provides satisfactory results when divalent immunoglobulins are used, provided one extracts the affinity only from the part of the saturation curve obtained at high saturation of the monoclonal antibody by the antigen.

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