IgE structure-function relationships defined by sequence directed antibodies induced by synthetic peptides.

Six peptides, representing contiguous amino acid sequences within the C epsilon 2, C epsilon 3 and C epsilon 4 domains of murine IgE, were selected for synthesis on the basis of overall hydropathy, degree of homology to both human and rat IgE and, where possible, inclusion of a native cysteine residue. Antibodies were produced against each peptide by immunizing rabbits with peptide-KLH conjugates. Each anti-peptide antiserum exhibited good reactivity with the corresponding immunizing peptide (titer: 10(-4) to 10(-5] and four of the six antisera exhibited a distinct preferential murine IgE reactivity compared to four other murine immunoglobulin classes (IgG1, IgG2b, IgM and IgA). In addition, one antiserum (anti-epsilon peptide 5), raised against a peptide with 80% homology to human IgE, reacted comparably with both human and murine IgE. Each IgE-reactive antiserum was screened for the ability to stimulate mediator release from IgE-sensitized rat basophilic leukemia (RBL) cells. Two of the four IgE-reactive antisera strongly stimulated 3H-serotonin release (anti-epsilon peptides 4 and 5), one antiserum showed weak activity (anti-epsilon peptide 3) and the remaining anti-peptide serum (anti-epsilon peptide 6), which exhibited the highest anti-IgE reactivity, exhibited no detectable stimulatory activity. Individual anti-peptide antibodies were subsequently tested for the potential to bind to receptor-bound IgE. Anti-epsilon peptide 3 was shown to exhibit the least binding, anti-epsilon peptide 6 showed the highest magnitude of binding while anti-epsilon peptides 4 and 5 exhibited intermediate values. We conclude from this study that sequences defined by epsilon-peptides 4 and 5 are not significantly involved in the receptor binding mechanism whereas epsilon-peptide 3 is likely to be most proximal to the IgE-receptor recognition site of those sequences studied. Finally, we suggest that the epsilon-peptide 6 sequence is in such an orientation in cell-bound IgE that, while it is accessible to external antibody, effective cross-linking of the IgE-receptor complex cannot be achieved through this determinant.