Use of a synthetic peptide epitope of Asp f 1, a major allergen or antigen of Aspergillus fumigatus, for improved immunodiagnosis of allergic bronchopulmonary aspergillosis

Abstract

Allergic bronchopulmonary aspergillosis (ABPA) is an immunologically complex allergic disorder caused by the fungal pathogen Aspergillus fumigatus. Elevated levels of total immunoglobulin E (IgE), specific IgE, and IgG antibodies in sera are important immunodiagnostic criteria for ABPA. International reference standards or standardized immunodiagnostic assays are not available due to a lack of well-defined diagnostic antigens. The present study was carried out to identify and evaluate the immunodiagnostic relevance of synthetic epitopic peptides of Asp f 1, a major allergen, antigen, or cytotoxin of A. fumigatus. Five overlapping peptides were synthesized from the N terminus of Asp f 1, one of the potential immunodominant regions predicted by algorithmic programs. The 11-amino-acid synthetic peptide (P1) significantly inhibited both IgG binding (89.10% +/- 4.45%) and IgE binding (77.32% +/- 3.38%) of the standardized diagnostic antigen (SDA) (a well-defined pool of diagnostically relevant allergens and antigens of A. fumigatus). With a panel of sera of ABPA patients, allergic patients with skin test negativity to A. fumigatus, and healthy individuals, P1 showed a higher diagnostic efficiency than SDA (specific IgG, 100%; specific IgE, 98.3%). The diagnostic efficiency of P1 could be attributed to the presence of homologous epitopes in various immunodominant allergens or antigens of A. fumigatus. The ability of P1 to induce histamine release from sensitized mast cells and a Th2 type of cytokine profile in peripheral blood mononuclear cells of ABPA patients suggests its potential for use in intradermal testing. P1 could be further explored for development of a standardized, specific, and sensitive immunodiagnostic test for aspergillosis.

FIG. 1.

Inhibition of binding of SDA to specific IgE and IgG antibodies in pooled sera of ABPA patients by the five synthetic peptides. To evaluate the diagnostic relevance of the synthetic peptides, the patient sera (diluted 1:100) were preincubated with various peptides (concentrations ranging from 50 ng to 1 μg). The immunoreactivities of the preincubated sera with SDA (1 μg/well) were analyzed by indirect ELISA. The inhibition percentages obtained with various peptides (100 ng/well) are presented here. The values represent the mean values of three readings, and the SD for each value was within a ±5% range.

FIG. 2.

Binding of peptide (80 ng/well by a dose-response curve) and SDA (1 μg/well) with specific IgE (A) and IgG (B) antibodies in sera of 20 clinically confirmed ABPA patients (ABPA), 20 controls (CTRL) (allergic patients with skin test negativity to A. fumigatus), and 25 healthy individuals (NRML) by indirect ELISA. The values represent the mean ELISA absorbance values for 20 ABPA patients, 20 controls, and 25 healthy individuals. The ELISA absorbance for each patient was the mean of three readings, and the SD for each value was in the ±5% range. The cutoff for each assay was calculated for SDA and P1 from the mean absorbance value obtained for the 20 healthy individuals plus 3 SD.

FIG. 3.

Cytokine levels in the PBMCs of ABPA patients incubated with SDA and P1. PBMCs of the ABPA patients and healthy individuals (Normals) were incubated with SDA, P1, and control peptide (Con Pep; HIV epitopic peptide) (each at a concentration of 10 μg/well). The supernatants were subjected to ELISA-based cytokine assays for human IFN-γ, IL-2, IL-4, IL-6, and IL-10. The values represent the mean values of three readings each from three ABPA patients and three healthy individuals, and the SD for each value was within a ±5% range.

FIG. 4.

P1-specific IgG and IgE antibody binding to electrophoresed allergens or antigens (SDA) on immunoblots (SDS-12% PAGE). Lane 1, molecular mass markers; lane 2, IgE binding of SDA with pooled sera of ABPA patients; lane 3, IgE binding of SDA with P1-specific antibodies; lane 4, IgG binding of SDA with pooled sera of ABPA patients; lane 5, IgG binding of SDA with P1-specific antibodies.