Isotypic analysis of grass pollen‐specific antibodies in human plasma. 4. Biological activity of allergen‐specific and autoanti‐IgE antibody fractions on basophil histamine release

Abstract

Blocking antibodies are defined as antibodies that compete with IgE for binding to allergens due to their specificity for those allergens. Thus, they may inhibit allergen-induced basophil and mast cell IgE-dependent mediator release both in vivo and in vitro. The present study was designed to evaluate the ability of antibodies isolated from human plasma samples on a Dactylis glomerata (Cocksfoot) pollen affinity-column to inhibit the Dactylis pollen-induced histamine release from human basophils (BHR) in vitro. Antibodies from Ig pools containing either high or low IgG4 anti-Dactylis pollen were purified on a Dactylis pollen affinity-column and then separated on an antihuman IgE column. Obtained Ig fractions were incubated for 30 min with Dactylis pollen allergens prior to incubation with basophils from Dactylis pollen-allergic donors. Cell supernatants were assessed for histamine content and the inhibition of BHR was calculated. Unlike control non-isolated Igs, the antibodies isolated on the Dactylis pollen column were able to inhibit efficiently and in a dose-dependent manner Dactylis pollen-induced BHR. The inhibitory activity was increased in isolated antibody samples that had high IgG4 levels. Antibodies isolated on the Dactylis pollen column, however, consisted not only of true allergen-specific (potentially blocking) antibodies but also of autoanti-IgE binding to allergen-specific IgE and mistaken for allergen-specific antibodies thus opening to question the involvement of the true allergen-specific antibodies in the BHR-inhibitory activity. Unlike the true allergen-specific antibodies, the autoanti-IgE were retained on and eluted from the anti-IgE column. Results showed that both the autoanti-IgE-depleted and the autoanti-IgE-containing fractions accounted for the inhibition observed with the related non-depleted sample that had been isolated on the Dactylis pollen column. For the first time, the true blocking activity of allergen-specific antibodies is demonstrated, that is, in the absence of the autoanti-IgE which can also inhibit BHR.