Primary and secondary immune response to human ige by rabbits immunized with synthetic ige peptides
Authors: Spiegelberg Hans L. , Canning Kathleen M. , Shields James E. , Chiller Jacques M. 
Published: March 2007

Rabbits were immunized with eight chemically synthesized peptides representing sequences of the third and fourth constant domains (Fc fragment) of human IgE to obtain antibodies to predetermined sites on IgE. The peptides, varying in length from 15 to 28 amino acids, were coupled to either keyhole limpet hemocyanin (KLH) or tetanus toxoid and injected in complete Freund's adjuvant. The resulting antisera were tested for binding to the peptides and IgE by a polyvinyl plate radioimmunoassay. Immunization with all eight peptides produced high titers of antibodies to the corresponding peptide, and six also induced antibodies that cross‐reacted with native IgE or IgE Fc fragments but not with IgE F(ab′)2 fragments or unrelated peptides and proteins. Conversely, goat, sheep, and rabbit antisera to native IgE or Fc IgE bound to the synthetic peptides that elicited cross‐reacting antibodies to IgE, indicative of a shared antigenic configuration between the peptides and IgE. However, the quantities of antipeptide antibodies cross‐reacting with IgE represented only a small fraction of the total antipeptide antibody titer, since absorption of the antisera with IgE reduced the titer of reactivity to the peptides only minimally and the yields of immunoadsorbent purified antibodies to IgE were low. Furthermore, the affinities of the antibodies cross‐reactive with IgE were low because the peptides inhibited binding to Fc IgE > 100 times more efficiently than did IgE. Additionally, the binding to Fc IgE of monovalent Fab fragments of purified antibodies cross‐reactive with IgE could no longer be demonstrated.

Because the synthetic peptides induced only low‐affinity cross‐reacting antibodies, the synthetic peptides were tested for the ability to prime rabbits for a secondary response to native IgE. Rabbits were injected with peptide‐KLH conjugates in complete Freund's adjuvant and challenged 6 months later with IgE in alum. None of the rabbits showed a secondary response to IgE; instead, they made a primary immune response identical to that of rabbits not given the synthetic peptides. Also, injection of IgE did not increase titers of antibody to the synthetic peptides still present 6 months after the primary injection, suggesting that the native IgE could not stimulate memory B cells induced by the peptide. Moreover, the nature of the secondary response to the synthetic peptide resembled that to a hapten in that it was elicited only by peptide conjugated to the carrier used for immunization but not to an unrelated carrier.

The data demonstrate that, although immunization of rabbits with synthetic peptides of the human ϵ chain coupled to KLH elicits the formation of antibodies cross‐reactive with native IgE, these rabbits do not become primed for a secondary response to IgE. This indicates that, before a synthetic peptide coupled to a highly immunogenic carrier is considered for use in a vaccine, its ability to induce a secondary response to the native protein must be established.