An Automated Method for Determination of Antibody Affinity Distribution Functions with Nanogram Quantities

We have developed a method to determine the binding affinity distribution functions (the probability density function for affinity constants will be referred to as affinity distribution throughout the text) for human serum antigen specific IgE for their respective allergens. This fully automated method, based on the Access technology of Sanofi Diagnostics Pasteur, is a highly sensitive two-step sandwich immunoassay requiring only nanogram quantities of antibodies. Allergen-antibody binding isotherms can be determined covering a range of 5 magnitudes of ligand (allergen) concentration. An affinity distribution function, describing the polyclonal nature of the antibody response, can be calculated from the binding isotherm. The validity of the method is assessed using mAbs against the purified allergen Der p 1. The resolving power and sensitivity of the method are demonstrated using selected sera from donors with high and low levels of specific IgE for these allergens. We are able to show that heterogeneous IgE populations can be determined using 100 microl of sera containing 150 pg of specific IgE per ml. Overall, the range of affinities that can be determined by this method is 1 x 10(6)-1 x 10(11) M(-1).