Since it has been shown that autoanti-IgE may be mistaken for antiallergen antibodies, thus appearing as pseudo-allergen-specific antibodies, it is crucial to separate true-from pseudo-allergen-specific antibodies and to determine to what extent autoanti-IgE appeared as pseudo-allergen-specific antibodies. For this purpose, human Ig pools were affinity-purified successively on a grass-pollen column and then on an antihuman-IgE column. IgG1-4, IgA, and IgM antibodies that were eluted from the grass-pollen column separated into pseudo- (approximately 30-40%) and true-allergen-specific antibodies that were coretained and not coretained, respectively, with the IgE on the anti-IgE column. Levels of autoanti-IgE were determined in individual plasma samples by surface plasmon resonance and statistically compared to the concentrations of allergen-specific antibodies obtained previously in the same plasma samples. A positive correlation between IgM autoanti-IgE levels and grass-pollen- "specific" IgM concentrations (P < 0.0002), and negative correlations between IgA autoanti-IgE and both IgE anti-grass pollen and IgG2 autoanti-IgE levels (P < 0.03, in both cases) were observed for the first time. This supports the contentions that: (1) autoanti-IgE antibodies appeared as pseudo-grass-pollen-specific antibodies, (2) they hid IgE antibodies when the latter were measured, and (3) they compete with one another in binding IgE. Lastly, a model of large Ig complexes is discussed.

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.

Previously, the specificity of human immune responses to Dactylis pollen was analyzed in 26 plasma samples with high levels of grass-pollen-specific IgG4 ('IgG4+ plasma', largely from grass-pollen-allergic patients), as compared to 25 plasma samples with low grass-pollen-specific IgG4 ('normal plasma', from nonatopic individuals). In the present study, a quantification of the Dactylis-pollen-specific IgE, IgM, IgA class and IgG subclass antibodies in these plasma samples is proposed. Isotypic distribution in IgG4+ plasma was 68% IgG [IgG2 (38%) > IgG4 (30%) > IgG1 (19%) > IgG3 (13%)], 27% IgM, 4% IgA and 0.05% IgE. In normal plasma it was 73% IgM, 20% IgG [IgG3 (38%) > IgG2 (33%) > IgG1 (29%) > IgG4 (0%)], 6% IgA and 0.006% IgE. In IgG4+ plasma, specific IgE, IgG1, IgG2 and IgG4 concentrations were positively correlated between each other. Finally, the present study clearly confirmed the possible role of the CH gene regulation in allergic diseases.