Sequence-specific antibodies against human IgE isoforms induced by an epitope display system.
BACKGROUND
Unlike other immunoglobulin isotypes, the human C epsilon gene generates by alternative splicing two types of secretory and two types of membrane epsilon chains. The two secreted epsilon heavy chains, epsilon(S1) and epsilon(S2), differ only in the sequence of the last eight C-terminal amino acids, being epsilon(S2) six amino acids longer. The two types of membrane isoforms differ in the extracellular membrane proximal domain, with the longer variant, epsilon(mL), containing 52 extra amino acids which are absent in the shorter epsilon(mS) isoform.


OBJECTIVE
We wished to produce quality antibody reagents that specifically detect epitopes that are epsilon isoform-specific.


METHODS
Short sequences of seven or ten amino acids were chosen as target epitopes and expressed as part of the highly immunogenic loops of deletion variants of engineered Flock House Virus capsid protein RNA2. Chimeric proteins were expressed in E. coli, and used to immunize rabbits. Antisera were screened by immunoblotting of purified IgE isoforms expressed by murine transfectomas.


RESULTS
Chimeric proteins expressing epsilon isoform-specific epitopes proved to be strong immunogens in vivo and induced highly specific rabbit antisera. Two antisera so obtained recognize specifically the IgE-S2 isoform. A third one recognizes the long membrane variant m(L)IgE and a fourth one detects an epitope specific to m(S)IgE.


CONCLUSIONS
Here we describe a simplified and efficient protocol of immunization which does not require peptide synthesis and conjugation to carrier protein. Our results show that short peptides of unknown immunogenicity, when genetically introduced into the modified Flock House Virus epitope display system, successfully induced IgE isoform-specific polyclonal antisera in rabbits. These are valuable tools to specifically identify secretory and membrane isoforms of human IgE, and the method is potentially applicable to other variant isoforms or mutants of a given protein.
Lorenzi
R
R
International Centre for Genetic Engineering and Biotechnology, Area Science Park, Trieste, Italy.
Burrone
O R
OR
D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000097:blood / Q000276:immunology*; D015153:Blotting, Western; D002213:Capsid / Q000235:genetics / Q000276:immunology; D000939:Epitopes / Q000276:immunology*; D005434:Flow Cytometry; D005803:Genes, Immunoglobulin; D006801:Humans; D007114:Immunization; D007073:Immunoglobulin E / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007139:Immunoglobulin epsilon-Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007304:Insect Viruses / Q000235:genetics; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D000888:Antibodies, Anti-Idiotypic; D000939:Epitopes; D007139:Immunoglobulin epsilon-Chains; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E