Antibodies Reactive with Native Lysozyme Elicited by a Completely Synthetic Antigen

Abstract

A synthetic peptide consisting of the aminoacid sequence of residues 64-82 of lysozyme, with alanine replacing cysteine as residue 76, was prepared by the solid-phase technique. Mild reduction followed by reoxidation in air of the deprotected peptide led to the formation of a closed loop containing an intrachain disulfide bond. A conjugate consisting of this "loop" attached to multi-poly(DL-alanyl)-poly(L-lysine) elicited, in rabbits and goats, the formation of antibodies capable of reacting with lysozyme and with the loop peptide prepared from it. These immunological interactions can be inhibited by either lysozyme or the loop peptide, but not by the performic acid-oxidized open-chain peptide. Thus, the antibodies elicited by the completely synthetic antigen show specificity toward the "loop" structure (residues 64-80) of native lysozyme.

Immunological cross-reactivity of antibodies to a synthetic undecapeptide analogous to the amino terminal segment of carcinoembryonic antigen, with the intact protein and with human sera.

“…A similar approach has been used for preparing specific antibodies towards peptides such as bradykinin (25), angiotensin (26), the tripeptide Tyr-Ala-Glu (27), the COOH-terminal hexapeptide of tobacco mosaic virus protein (28), and towards a synthetic peptide, the "loop", comprising the residues 64-82 in the amino LI. acid sequence of lysozyme (29). In all these cases the specific antibodies recognized the moiety present in the immunogen, but not necessarily a larger structure of which they form a part.…”

“…Similarly, antibodies to the COOH-terminal hexapeptide of tobacco mosaic virus protein do not react with the intact tobacco mosaic virus protein (30). On the other hand, antibodies to the synthetic loop fragment of lysozyme are fully reactive with the intact native molecule (29). These differences are due to the presence of both sequential and conformational antigenic determinants on macromolecules (27), and are therefore determined by the conformation of the isolated or synthetic fragment as compared to the conformation it assumes in the intact macromolecule or larger molecular structure.…”

Mapping epitopes and antigenicity by site-directed masking

“…4B) is due to the presence of antibodies raised against denatured antigen that crossreact with exposed and flexible segments. It is well established that immunization with peptide segments comprising flexible loop regions of the antigen can elicit antibodies reactive to the native state of the antigen (27).…”

From Proteins and Protein Models to Their Use in Immunology and Immunotherapy

“…In addition, we could demonstrate for the first time, by circular dichroism, how antibodies to the ␣-helical polymer could help transconform into a helical shape a small polymer that was not yet helical (32). These studies led us directly to study proteins and to synthesize a macromolecule in which a synthetic "loop" peptide derived from hen egg white lysozyme was attached to branched polyalanine (33). The resulting antibodies reacted with intact lysozyme through the "loop" region, but the reaction was totally abolished when the disulfide bond within the "loop" was opened, and thus the three-dimensional structure was collapsed.…”