Background: The venomous stings of Jack Jumper ant (JJA; species of the Myrmecia pilosula taxonomic group) are a significant public health issue in parts of south‐eastern and south‐western Australia, causing anaphylaxis in approximately 3% of the population. Three allergenic peptides, Myr p 1, Myr p 2 and Myr p 3, and one histamine‐releasing peptide, pilosulin 5, have been fully described, but there are at least 5 additional high molecular weight IgE‐binding components that have not been identified.

Objective: To identify IgE‐binding components in JJA venom (JJAV) and to relate the IgE recognition of these components to relevant clinical parameters.

Methods: Identification of IgE‐binding components and determination of their sensitizing prevalence was performed using SDS‐PAGE immunoblot assay and sera from 90 patients with confirmed allergy to JJAV. Tandem mass spectrometry was used for identification of novel JJAV components fractionated by size exclusion chromatography (SEC) and SDS‐PAGE.

Results: Using SDS‐PAGE immunoblot, 10 IgE‐binding bands were identified in JJAV, two of which were recognized by 81% and 47% of the population studied. Mass spectrometry identified 17 novel JJAV proteins, including 2 glycoproteins, and confirmed the presence of 4 known Myr p and pilosulin peptides in JJAV. Most of the newly identified IgE‐binding proteins were enzymes, including phospholipase A2, hyaluronidase, arginine kinase and dipeptidyl peptidase IV. Correlations were found between recognition of certain IgE‐binding bands with JJAV‐specific IgE titre by ImmunoCAP, intradermal test threshold and treatment‐related issues.

Conclusions and Clinical Relevance: This study has for the first time revealed the identity of various proteins with IgE‐binding capacity in the venom of JJA and demonstrated their clinical relevance in the diagnosis and treatment of JJAV allergy.