Absolute quantitation of serum antibody reactivity using the Richards growth model for antigen microspot titration

Abstract

In spite of its pivotal role in the characterization of humoral immunity, there is no accepted method for the absolute quantitation of antigen specific serum antibodies.We devised a novel method to quantify polyclonal antibody reactivity, which exploits protein microspot assays and employs a novel analytical approach. Microarrays with a density series of disease specific antigen were treated with different serum dilutions and developed for IgG and IgA binding. By fitting binding data of both dilution series to a product of two generalized logistic functions, we obtained estimates of antibody reactivity of two immunoglobulin classes simultaneously. These estimates are the antigen concentrations required for reaching the inflection point of thermodynamic activity coefficient of antibodies and the limiting activity coefficient of antigen.By providing universal chemical units, this method may improve standardization of serological testing, the quality control of antibodies and the quantitative mapping of antibody-antigen interaction space.

The Binding Landscape of Serum Antibodies: How Physical and Mathematical Concepts can Advance Systems Immunology

“…The rate of change in this quantity, while changing reaction conditions by titrating antibody or antigen, is the derivative of the function that relates the amount of complex formed to the logarithm of the titrated component. We proposed the use of the generalized logistic model or Richards growth model [20,21] instead of 5PL [22], because the Richards growth function, like the 5PL, is the solution of the differential equation…”

“…One of these is the antigen concentration at the point of inflection [Ag]i and the other is the limiting activity coefficient (Figure 4.) [22]. The first is an estimate of the apparent equilibrium dissociation constant (determined by average standard chemical potential) of the antibodies bound to the Ag.…”