Icarapin Antibody

What is icarapin and what are its general characteristics?

Icarapin, officially designated as Api m 10, is a 204 amino acid protein found in honeybee venom (Apis mellifera). It contains 2-4 potential N-linked glycosylation sites and is characterized by its instability and low abundance in the venom . The protein was first identified after 2D-SDS-PAGE separation of pure honeybee venom followed by mass spectrometry analysis of excised spots, initially referred to as "new venom protein 2" . It is located in the cuticular lining of the venom duct of the honeybee, with minor presence in secretory cells . The name "icarapin" is an artificial term combining "Icarus" from Greek mythology and the genus name "Apis," reflecting its unstable nature and rapid degradation .

How prevalent is sensitization to icarapin in honeybee venom-allergic patients?

Sensitization rates to icarapin vary across different studies, as summarized in the table below:

These variations may be attributed to differences in patient inclusion criteria and geographical differences in sensitization profiles. Despite being a protein of low abundance in honeybee venom, icarapin is recognized as a major allergen due to the significant proportion of patients (35-72%) exhibiting IgE reactivity to it .

What is the role of icarapin in component-resolved diagnostics (CRD)?

Icarapin serves as a valuable marker allergen for primary honeybee venom sensitization, helping to discriminate between genuine sensitization and cross-reactivity . In a study of double-sensitized patients (to both honeybee and yellow jacket venom), the addition of Api m 10 testing significantly improved diagnostic accuracy. Among double-sensitized patients negative for Api m 1, sIgE to Api m 10 was found in 29% of cases, confirming primary sensitization to honeybee venom that would have been missed using conventional diagnostics .

When used in combination with other allergens, diagnostic sensitivity improves substantially. For instance, adding Api m 10 to Api m 1 testing increased diagnostic sensitivity from 72% to 87% in one study, while a combination of six allergens (Api m 1-5 and 10) achieved 94% sensitivity .

What are the known IgE epitopes of icarapin?

Recent research using macroarray platforms coated with 15-mer peptides (with 12-amino acid overlaps) spanning the entire amino acid sequence of mature icarapin has identified key epitope regions. All tested sera from Api m 10-positive patients (n=40) exhibited sIgE reactivity that correlated with values obtained by ImmunoCAP testing .

Individual patient sera recognized up to 29 different peptides, with three regions showing IgE binding in more than 40% of the sera. Most notably, one peptide (Api m 10160–174; amino acid sequence: ADSDVTTLPTLIGKN) was recognized by 100% of the Api m 10-sensitized patients and exhibited higher sIgE reactivity than any other peptide . This finding has potential implications for diagnostic test development and has led to a patent application (pending registration number EP19199738) .

How does icarapin contribute to the efficacy of venom immunotherapy (VIT)?

Emerging evidence suggests that sensitization to icarapin may influence VIT outcomes. Dominant sensitization to Api m 10 has been identified as a risk factor for treatment failure, which is particularly significant since Api m 10 is strongly underrepresented in some therapeutic preparations commonly used for VIT .

This underrepresentation might explain treatment failures in certain patients. In one study analyzing the composition of different HBV VIT preparations, some products showed substantial underrepresentation of Api m 10 compared to crude venom, while others contained detectable amounts . This finding highlights the importance of considering individual sensitization profiles when selecting therapeutic preparations for VIT.

What approaches can reduce icarapin antigenicity for therapeutic applications?

Research has explored methods to reduce icarapin antigenicity, which could be valuable for improving VIT outcomes. In a study of Asian honeybee icarapin, in silico screening identified S198 as a potential antigenic site . Researchers changed this site to cysteine and coupled it with polyethylene glycol (PEG5K) .

The PEGylated S198C variant induced lower levels of IgG and IgE antibodies in mice compared to both wild-type icarapin and the unmodified S198C variant, confirming that this site is indeed located in an antigenic region . This approach of site-specific PEGylation represents a potential strategy for reducing allergenicity while maintaining therapeutic efficacy.

What techniques are used for recombinant production of icarapin?

Various expression systems have been employed for recombinant icarapin production, each with distinct advantages:

• E. coli production as insoluble protein: Early attempts produced icarapin as an insoluble protein in E. coli, which confirmed IgE binding capability but had limitations for functional studies .

• E. coli production as soluble protein: Improved methods later achieved soluble expression in E. coli, facilitating more detailed immunological studies .

• Eukaryotic insect cell expression: This system, used by Blank et al., produces properly folded and post-translationally modified icarapin, allowing for more physiologically relevant studies of IgE reactivity and basophil activation tests .

The choice of expression system depends on the research goals, with insect cell expression providing more native-like proteins suitable for diagnostic and therapeutic applications, while bacterial systems may offer higher yields for structural studies.

How is icarapin sensitization detected in clinical research?

Several methodologies have been employed to detect and characterize icarapin sensitization:

• ImmunoCAP assays: Commercially available since 2015, these assays use recombinant Api m 10 to quantitatively measure specific IgE (sIgE) with a detection threshold typically set at ≥0.35 kUA/L . This platform allows standardized testing across different laboratories.

• Basophil activation test (BAT): This cellular assay measures the activation of basophils upon exposure to allergens, providing functional confirmation of sensitization. In one study, the basophils of 62% (8/13) of honeybee venom-allergic patients were activated in a dose-dependent manner by Api m 10 .

• Peptide macroarray analysis: This technique utilizes synthetic overlapping peptides spanning the entire sequence of Api m 10 to identify specific IgE epitopes, providing detailed information about the regions of the protein most relevant for allergic sensitization .

How can researchers assess cross-reactivity between icarapin and homologous proteins?

Cross-reactivity assessment is critical for understanding the specificity of allergic responses and improving diagnostic accuracy. Several approaches can be employed:

• Sequence alignment analysis: Computational comparison of amino acid sequences can identify potential regions of homology. For example, analysis has revealed homologs of icarapin in other insect species, including paper wasp venom (PDV icarapin) .

• Inhibition studies: Pre-incubation of patient sera with one allergen followed by testing reactivity to another can reveal cross-inhibition, indicating shared epitopes.

• Epitope mapping: Identifying and comparing specific epitopes between different allergens can provide insights into the molecular basis of cross-reactivity. Importantly, the major IgE epitope identified in Api m 10 is not present in its paper wasp venom homolog, which may explain the minimal cross-reactivity observed between these proteins .

What are the priority areas for advancing icarapin research?

Based on current knowledge gaps, several research priorities emerge:

• Large international multicenter studies: These are needed to assess the clinical relevance of icarapin sensitization across different geographical regions and patient populations .

• Improved therapeutic preparations: Development of VIT preparations with standardized and appropriate amounts of all clinically relevant allergens, including icarapin .

• Structure-function studies: Further elucidation of the biological function of icarapin, which remains largely unknown, could provide insights into its role in the venom and potential therapeutic applications.

• Personalized treatment approaches: Investigation of how individual sensitization profiles, particularly to icarapin, can inform personalized risk stratification and treatment selection for HBV-allergic patients .