Determination of the concentration of influenza virus antihaemagglutinin antibody molecules of the IgG class and of the equilibrium constant by use of enzyme immunoassay titres determined for graded epitope concentrations.

A new technique for determining the concentration of influenza virus antihaemagglutinin antibody molecules of the IgG class (A) and of the equilibrium constant K of paratope-epitope interaction is described. The method is based on determining enzyme immunoassay (EIA) titres of antibody with graded epitope concentrations: EIA titres were defined in terms of the antibody dilution yielding a fixed amount of antibody adsorbed per ml (= 6.21 x 10(10)). Adsorption of antibody depends on the concentration of paratopes and epitopes allowed to react and on the equilibrium constant. For the use of a constant concentration of epitopes, the paratope concentration needed to yield the desired degree of antibody adsorption decreases with increasing avidity. Therefore, the EIA titres increase both with increasing avidity and increasing antibody concentration. When graded epitope concentrations are used for determining the EIA titres of a given serum, the titres are influenced in a similar manner by the antibody concentration of the serum and the increase of titres with increasing epitope concentration reflects avidity. The equilibrium constant found is subsequently used to determine the concentration of free antibody at the dilution meeting the definition of EIA titre and the product of EIA titre and the sum of free and bound antibody at this dilution gives the number of antibody molecules present in the test serum. A panel of 118 antisera was tested comparatively for A and K using the new method and by means of the guanidine titre ratio test and equilibrium filtration. The values obtained agreed well with each other. This novel technique offers the advantage that it can be easily adapted for use with other viruses.