Isolation of an immunodominant IgE hapten from an epitope expression cDNA library. Dissection of the allergic effector reaction.

An epitope expression cDNA library was constructed from the randomly fragmented cDNA coding for Phl p I, the major grass pollen allergen. Using IgE from allergic patients, epitope clones were isolated and immunodominant fragments were selected. Among three epitope clones coding for a similar region of Phl p I, one clone expressed a 15-amino-acid epitope which was target for IgE antibodies from approximately 30% of grass pollen allergic patients. According to the prevalence of grass pollen allergy, 22% of all allergic patients are expected to display IgE reactivity with this epitope. Although the purified recombinant epitope specifically bound IgE, it did not release histamine from basophiles of most grass pollen allergic patients and thus represents an IgE hapten. Immunodominant IgE haptens may be useful as therapeutic agents to saturate mast cell-bound IgE prior to allergen exposure and may represent candidates for a safe immunotherapy of allergic diseases by reducing anaphylactic side effects.

Allergens of Parietaria judaica pollen--I. Purification and characterization of a hapten and a low molecular weight allergenic peptide.

A low mol. wt allergen (Pj-2) and a hapten (Pj-H3) were purified from Parietaria judaica pollen by means of long-term aqueous extraction, dialysis and gel filtrations. The yield of the Pj-2 allergen was 0.94% (w/v) of the total protein present in the aqueous extract of the pollen, while its allergenic activity was about 60% of the total dialyzable activity, as verified by skin prick tests, ELISA- and RAST-inhibition experiments. The homogeneity of this allergen was demonstrated by one single sharp peak on HPLC, one single band on PAGE-SDS and by one single arc on IEF. Its mol. wt, estimated by HPLC and amino acid composition, was 10,400. The amino acid analysis showed 73 amino acid residues, and lysine was predominant, with 20 residues. The hapten Pj-H3 was 0.2% (w/v) of the total protein found in the pollen aqueous extract. It was inactive in skin prick tests even at a protein concn of 2 mg/ml, while it was capable of inhibiting by 60% in ELISA- and RAST-inhibition experiments, suggesting an immunochemical relationship with both IgE and allergens specific to P. judaica. The homogeneity was demonstrated by one single sharp peak on HPLC and one single band on PAGE-SDS. The amino acid analysis showed 10 amino acid residues, with no specific traits, and the mol. wt determined by gel filtration and amino acid composition was 1000. An immunochemical relation between the allergen and the hapten was also suggested by the results of an ELISA-inhibition test, and by the ability of the hapten to partially inhibit the precipitin line between rabbit antibodies to whole P. judaica pollen extract and the Pj-2 allergen. The allergen and the hapten described above, purified at homogeneity and in an antigenically active state, both provide adequate material for further structural and immunological characterizations.

Allergic contact dermatitis to laurel (Laurus nobilis L.): isolation and identification of haptens.

Two methods, using methylenelactone dimethylamino adducts, were used to remove selectively alpha-methylene gamma-butyrolactones from Laurus nobilis L. extracts. Isolated lactones were identified and "treated" extracts recovered. Two guinea-pig groups were sensitized to crude extracts and "treated" extracts, respectively, and tested with primary sensitizer and with different lactones. Only the first group showed strong skin reactions to crude extracts and to the lactones. Treated extracts were shown to be anallergic.

Allergic contact dermatitis to costus: removal of haptens with polymers.

Costus oil, an iol isolated from a Composite, Saussurea lappa C., used in perfumery, is responsible of numerous cases of allergic contact dermatitis (ACD). The haptens incriminated are sesquiterpene lactones, costunolide and dehydrocostuslactone. These lactones can be removed from the oil by using a polymer, aminoethyl-polystyrene, to which they become bound: the passing solution is lactone-free. A group of guinea-pigs could be successfully sensitized (Freund Complete Adjuvant Test) to costus essential oil, while another group, injected in the same way with polymer-treated essential oil (i.e. lactones) could not be sensitized. This polymer therefore provides a means to render complex plant extracts non-allergic.

Allergens of Kentucky Blue Grass pollen. II. Isolation of hapten-like components from Kentucky Blue Grass pollen by preparative isoelectrofocussing.

Components with hapten-like properties were isolated from the nondialyzable fraction, i.e. the retentate (R) and the dialyzable fractions of the aqueous extract of Kentucky Blue Grass pollen (KBC aq.ext.), by preparative isoelectrofocussing on Sephadex G-100 gel. These haptenic components could not elicit the passive cutaneous anaphylaxis (PCA) reactions in rats passively sensitized with a murine reaginic antiserum to R, but could inhibit completely and specifically the PCA reaction which is normally elicitable with R. It was concluded that the specificity of the murine IgE antibodies was directed to a determinant(s) which was common to either allergenic or haptenic fractions. Moreover, by employing a pool of human sera from individuals allergic to KBG pollen in the radioallergosorbent test procedure, it was apparent that most of the haptenic fractions lacked some of the specificities present on allergenic components of R that were recognized by the human IgE antibodies. Evidence was obtained to suggest that the electrophoretic heterogeneity of allergenic components present in various fractions of KBG aq.ext. may be due primarily to differences in their net charge, rather than to differences in their allergenic specificity.