Allergen-specific IgG antibodies purified from mite-allergic patients sera block the IgE recognition of Dermatophagoides pteronyssinus antigens: an in vitro study

Abstract

One of the purposes of specific immunotherapy (SIT) is to modulate humoral immune response against allergens with significant increases in allergen-specific IgG levels, commonly associated with blocking activity. The present study investigated in vitro blocking activity of allergen-specific IgG antibodies on IgE reactivity to Dermatophagoides pteronyssinus (Dpt) in sera from atopic patients. Dpt-specific IgG antibodies were purified by ammonium sulfate precipitation followed by protein-G affinity chromatography. Purity was checked by SDS-PAGE and immunoreactivity by slot-blot and immunoblot assays. The blocking activity was evaluated by inhibition ELISA. The electrophoretic profile of the ammonium sulfate precipitated fraction showed strongly stained bands in ligand fraction after chromatography, compatible with molecular weight of human whole IgG molecule. The purity degree was confirmed by detecting strong immunoreactivity to IgG, negligible to IgA, and no reactivity to IgE and IgM. Dpt-specific IgG fraction was capable of significantly reducing levels of IgE anti-Dpt, resulting in 35%-51% inhibition of IgE reactivity to Dpt in atopic patients sera. This study showed that allergen-specific IgG antibodies purified from mite-allergic patients sera block the IgE recognition of Dermatophagoides pteronyssinus antigens. This approach reinforces that intermittent measurement of serum allergen-specific IgG antibodies will be an important objective laboratorial parameter that will help specialists to follow their patients under SIT.

Figure 1

(a) Levels of IgE, IgG1, and IgG4 antibodies to Dermatophagoides pteronyssinus (Dpt) allergen extract in serum samples from atopic and nonatopic patients. Data are expressed in ELISA index (EI), and mean is indicated by horizontal bars. The dashed line indicates the cutoff of the reaction (EI > 1.2). Percentages of positive samples are also indicated. Statistically significant differences were determined by the Mann-Whitney test (*P < 0.05; ***P < 0.0001). (b) Correlation between levels of Dpt-specific IgE versus IgG1 and IgE versus IgG4 anti-Dpt in serum samples from atopic patients. Percentages of double positive, double negative, or single positive for each antibody class are indicated in the correspondent corners. Spearman's correlation coefficient and statistical significance are also indicated. (c)  Comparison between levels of IgG1 and IgG4 anti-Dpt in serum samples from atopic and non-atopic patients. Five serum samples (red ellipses) of each patient group were selected to constitute the specific (atopic) and nonspecific (nonatopic) total IgG pools. The dashed lines indicate the cutoff of the reaction (EI > 1.2).

Figure 2

(a) Slot-blots showing reactivity for IgE, IgA, IgG, and IgM in the serum, supernatant (S 40%), and precipitated (P 40%) fractions obtained from precipitation of the serum with 40% ammonium sulfate solution, and bovine serum albumin (BSA) as irrelevant protein. (b) Slot-blot data analysis by measuring the intensity of the bands in gray scale and expressed in arbitrary units (AU). Statistically significant differences were determined by Student's t test (**P < 0.01; **P < 0.001).

Figure 3

(a) Representative chromatogram of the total human IgG purification by affinity chromatography in protein G-agarose obtained from the 40% ammonium sulfate precipitated fraction (P 40%) of a serum pool. I—elution peak of P 40% nonligand fraction after washing with 0.02 M phosphate buffer pH 8.0 (black arrow); II—elution peak of P 40% ligand fraction after washing with 0.1 M glycine buffer pH 2.6 (dashed arrow). Data are expressed in absorbance (280 nm). Elution volume consisted of 1 mL in each tube. Values of pH were also measured in each elution tube. (b) Electrophoretic profile in SDS-PAGE 8% stained with blue silver. NLF—nonligand fraction, LF—ligand fraction, corresponding to the tubes 29–34. Markers of molecular weight (MW) are indicated on the left in kilodaltons (kDa). (c) Immunoblots for detection of IgG, IgA, IgE, and IgM in the serum fractions after purification in protein G-Agarose as shown in (b). Bands were revealed with DAB as described in Methods.

Figure 4

Levels of IgG1 (a), IgG4 (b), and IgE (c) antibodies to Dermatophagoides pteronyssinus (Dpt) allergen extract in the specific (Sp) and nonspecific (Ns) purified IgG fractions obtained from atopic and nonatopic patients, respectively, determined by ELISA. Purified IgG fractions were titrated at two-fold dilutions from 80 to 2.5 µg/well, and data are expressed in ELISA index (EI). The dashed lines indicate the cutoff of the reaction (EI > 1.2). The values indicating the Sp/Ns ratio for each antibody class and analyzed concentration are also indicated.

Figure 5

Inhibition ELISA results showing the blocking capacity of specific and nonspecific purified IgG antibodies for IgE reactivity to Dermatophagoides pteronyssinus (Dpt) allergen extract in serum pools of atopic patients. Three serum pools (I, II, and III) with different positivity for antibody classes were used as follows: pool I (IgE+, IgG1+, and IgG4−); pool II (IgE+, IgG1−, and IgG4+); and pool III (IgE+, IgG1+, and IgG4+). (a) Levels of IgE anti-Dpt expressed in absorbance (405 nm). Statistically significant differences were determined by one-way ANOVA and the Bonferroni posttest (***P < 0.0001). (b) Percentage of inhibition of IgE binding by blocking specific and nonspecific IgG antibodies in three serum pools of atopic patients. The dashed line indicates a threshold inhibition value of 35%. Statistically significant differences were determined by Student's t-test (**P < 0.01; ***P < 0.0001).