Analysis of the effector activity of Ara h 2 and Ara h 6 by selective depletion from a crude peanut extract

Abstract

It is important to know the contribution of specific allergens to a complex allergenic extract and to have a dependable method to assess the effector activity of an extract specifically depleted of that allergen. We have previously shown that removal of the major peanut allergen, Ara h 2, from a crude peanut extract (CPE) minimally altered the effector activity of the extract. Here we describe in detail the methodology used to generate specific rabbit anti-peptide antibodies to remove a related peanut allergen, Ara h 6, from CPE and describe an improvement in the RBL SX-38 cell assay used to assess the effector activity of treated extracts. Our results show that although Ara h 2 and Ara h 6 can be selectively removed from a CPE, removal of each alone from a CPE had no significant effect on the effector activity. However, removal of Ara h 2 and Ara h 6 together significantly reduced the effector activity of CPE.

Figure 1. Kyte-Doolittle plot of Ara h 6 showing the hydrophilicity, surface probability and antigenic index over the length of the Ara h 6 amino acid sequence

The candidate regions for which we made anti-peptide antibodies that were not able to immunodeplete Ara h 6 are underlined and the peptide that was used to produce the antibody that specifically depleted of Ara h 6 is enclosed in a box.

Figure 2. Immunoblot of CPE following addition of increasing amounts of CPE over a column with affinity purified rabbit anti-Ara h 6 (peptide, EQEQYDSYDIRSTRSSDQ)

Lane 1, untreated CPE (not passed over a column). Lanes 2–8, 1.5 ml of CPE at various concentrations as shown were passed over 1 ml of packed beads containing 4 – 4.5 mg of antibody. Beads were linked to the rabbit anti-Ara h 6 antibody for lanes 2, 3, 4, 6, 7, and 8. For lanes 5 and 9 the beads were linked to pre-immune IgG. CPE at 3 mg/ml (4.5 mg total) passed over an anti-Ara h 6 columns is shown in lane 2 (one pass) and lane 6 (two passes). CPE at 6 mg/ml (9 mg) passed over an anti-Ara h 6 column is shown in lane 3 (one pass) and lane 7 (two passes). CPE at 12 mg/ml (18 mg) passed over an anti-Ara h 6 column is shown in lane 4 (one pass) and lane 8 (two passes). The immunoblot was probed with a mixture of antibodies to Ara h 1, 2, and 6 as described in the materials and methods section.

Figure 3. Immunodepletion of Ara h 2, Ara h 6, and both

Immunoblots are shown probed with a combination of rabbit anti-Ara h 1, 2, and 6 antibodies and developed with anti-rabbit IgG (A) and probed with the human pooled serum developed with anti-human IgE (B). Each lane contained 1µg (A) or 2 µg (B) of CPE following passage over columns containing both anti-Ara h 2 and anti-Ara h 6 (lane 1), anti-Ara h 6 alone (lane 2), anti-Ara h 2 alone (lane 3), or pre-immune IgG (lane 4). For this figure, the ratio of CPE to packed beads was ~4.5 mg of CPE to 1 ml of packed beads containing ~4 mg of affinity purified antibody.

Figure 4. Effector activity of CPE

RBL-AB cells were sensitized with IgE from a pool of sera from highly peanut-allergic subjects as described in the materials and methods section and stimulated with CPE depleted of Ara h 2 alone (A), depleted of Ara h 6 alone (B) and depleted of both Ara h 2 plus Ara h 6 (C) as shown in Figure 3. Solid symbols are the CPE treated with pre-immune serum and open symbols are allergen depleted CPE. In each experiment (A, n=3; B, n=3; C, n=7), cells were stimulated in triplicate with a maximal amount of CPE (200 mg/ml). In order to pool the experimental results, the data are expressed as a percent of the maximal net (total minus background) degranulation in each assay. Maximal net degranulation with CPE (mean±SEM): A, 41±5%; B, 42±4%; C, 40±2%. If the standard error bars are not visible, they were smaller than the symbol. Best-fit lines were generated using a one-phase decay model (Prism 5.0c for MacIntosh; GraphPad Software, Inc. La Jolla, CA).

Figure 4. Effector activity of CPE

RBL-AB cells were sensitized with IgE from a pool of sera from highly peanut-allergic subjects as described in the materials and methods section and stimulated with CPE depleted of Ara h 2 alone (A), depleted of Ara h 6 alone (B) and depleted of both Ara h 2 plus Ara h 6 (C) as shown in Figure 3. Solid symbols are the CPE treated with pre-immune serum and open symbols are allergen depleted CPE. In each experiment (A, n=3; B, n=3; C, n=7), cells were stimulated in triplicate with a maximal amount of CPE (200 mg/ml). In order to pool the experimental results, the data are expressed as a percent of the maximal net (total minus background) degranulation in each assay. Maximal net degranulation with CPE (mean±SEM): A, 41±5%; B, 42±4%; C, 40±2%. If the standard error bars are not visible, they were smaller than the symbol. Best-fit lines were generated using a one-phase decay model (Prism 5.0c for MacIntosh; GraphPad Software, Inc. La Jolla, CA).

Figure 4. Effector activity of CPE

RBL-AB cells were sensitized with IgE from a pool of sera from highly peanut-allergic subjects as described in the materials and methods section and stimulated with CPE depleted of Ara h 2 alone (A), depleted of Ara h 6 alone (B) and depleted of both Ara h 2 plus Ara h 6 (C) as shown in Figure 3. Solid symbols are the CPE treated with pre-immune serum and open symbols are allergen depleted CPE. In each experiment (A, n=3; B, n=3; C, n=7), cells were stimulated in triplicate with a maximal amount of CPE (200 mg/ml). In order to pool the experimental results, the data are expressed as a percent of the maximal net (total minus background) degranulation in each assay. Maximal net degranulation with CPE (mean±SEM): A, 41±5%; B, 42±4%; C, 40±2%. If the standard error bars are not visible, they were smaller than the symbol. Best-fit lines were generated using a one-phase decay model (Prism 5.0c for MacIntosh; GraphPad Software, Inc. La Jolla, CA).