Assessment of IgG avidity against pertussis toxin and filamentous hemagglutinin via an adapted enzyme-linked immunosorbent assay (ELISA) using ammonium thiocyanate

Abstract

Antibody avidity, defined as the strength of binding between antibody and antigen, represents a functional measure of affinity maturation of antibodies. Determination of the antibody avidity is usually performed separating high and low avidity antibodies by dissociating agents, but measurement of the antibody avidity in humans is rather complicated, due to the heterogeneity of the antibodies produced in response to complex antigens, e.g. after vaccinations.The purpose of the present study was to evaluate the experimental determinants of the assessment of avidities of IgG antibodies directed against pertussis toxin (IgG-anti-PT) and filamentous hemagglutinin (IgG-anti-FHA) produced after pertussis vaccination using an adapted ELISA and ammonium thiocyanate (NH4SCN) as dissociating agent.Our experiments revealed that the results of avidity testing depend very much on experimental conditions and may over- or underestimate the relative avidity of IgG-anti-PT and IgG-anti-FHA antibodies. Whereas in our findings avidity seems to be independent from the initial antibody concentration in a wide range of measures, RAI depends on NH4SCN concentration, time of incubation and temperature of the reaction.The presented method allows an accurate measurement of the IgG antibody avidity against both Bordetella pertussis antigens PT and FHA, using NH4SCN as chaotropic agent in concentrations lower than 3.0M for 20min time of incubation at 37°C. Different experimental conditions in testing pertussis-specific IgG antibody avidity should be considered in interpretation and comparability of data of different studies.