Background: 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.
Objective: 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.
Methods: 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.
Results: 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.
Conclusion: 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.