Sequence-specific antibodies against human IgE isoforms induced by an epitope display system

Area of Science:

• Immunology
• Molecular Biology
• Protein Engineering

Background:

• The human C epsilon gene produces four IgE isoforms through alternative splicing: two secretory (epsilon(S1), epsilon(S2)) and two membrane-bound (epsilon(mL), epsilon(mS)).
• These IgE isoforms differ in their C-terminal sequences or extracellular domains, presenting unique epitopes.

Purpose of the Study:

• To generate high-quality antibody reagents that specifically detect epsilon isoform-specific epitopes.
• To develop a simplified and efficient immunization protocol for producing such antibodies.

Main Methods:

• Short peptide sequences (7-10 amino acids) representing IgE isoform-specific epitopes were genetically fused to engineered Flock House Virus capsid protein RNA2.
• Chimeric proteins were expressed in E. coli and used to immunize rabbits, with antisera screened against purified IgE isoforms.

Main Results:

• The chimeric proteins acted as potent immunogens, inducing specific rabbit antisera.
• Four antisera were successfully generated, with two specifically recognizing the IgE-S2 isoform, one targeting the m(L)IgE variant, and another detecting the m(S)IgE isoform.

Conclusions:

• A simplified, efficient immunization protocol was established, eliminating the need for peptide synthesis and carrier conjugation.
• Genetically incorporating short peptides into the Flock House Virus epitope display system successfully generated IgE isoform-specific polyclonal antisera.
• These antibodies are valuable tools for identifying human IgE secretory and membrane isoforms and the method is adaptable for other protein variants.