Calculation of the affinity constant KASS for solid phase antigen : A model system using monoclonal antibodies against the cat allergen Fel d I

Abstract

In this report a procedure is described to calculate the affinity constant of an antibody for solid-phase Ag. K SP ( K ASS solid phase) was defined as the reciprocal of the concentration of Ag required for half saturation of the Ab-binding sites, extrapolated to an infinitely small concentration of Ab (semi-saturation plot). Using this procedure, the affinity of IgE antibodies can be measured without interference from ‘invisible’ IgG antibodies. As a model system, mAbs against the cat allergen Fel d I were used. Serial dilutions of Fel d I-Sepharose were incubated with serial dilutions of mAb Fd1a and Fd1b, with or without rabbit antibodies as ‘invisible’ antibodies. The Ab-binding capacity of Sepharose-coupled Fel d I was low: 4.15% and 2.13% of the expected value for mAb Fd1a and Fd1b, respectively, and this must be taken into account when calculating K SP. The K values of mAb Fd1a and Fd1b, calculated from the y axis intercept of the semi-saturation plot, were 85 (pmol/test) −1 and 65 (pmol/test) −1 respectively. Using the semi-saturation plot, K SP of mAb Fd1a was not affected by the presence of rabbit antibodies against Fel d I, confirming the applicability of the procedure for measuring the K SP of IgE in patient sera. For one cat-allergic patient the K SP of IgE and IgG4 for Fel d I were calculated and found to be 62 (pmol/test) −1 and 147 (pmol/test) −1 for IgE and IgG4 respectively.