Peptides as antigens. Importance of orientation.

Abstract

Factors known to be important in producing protein-reactive peptide antibodies include the accessibility of the region from which the peptide sequence is derived, the hydrophilic-phobic character of the sequence, and the length of the peptide. The data presented here indicate that the orientation of the peptide coupled to a carrier protein also influences the binding pattern of peptide antibodies. An octapeptide, representing a sequence from the alpha chain of the human acetylcholine receptor, was coupled either through an N- or C-terminal cysteine-glycine-glycine linker to a carrier protein and used to immunize rabbits. The resulting antisera reacted at comparable titers to the uncoupled immunizing peptides, but did not crossreact with the identical but opposite-linked peptide. Characterization of the binding to other homologous peptides showed that immunization with the N-terminal-linked peptide induced antibodies reactive specifically with the C-terminal amino acid(s). Immunization with the C-linked peptide resulted in antibodies reactive with a site of the peptide near the C-terminus.

Synthetic peptide vaccine confers protection against murine malaria.

“…The reasons for this are not clear except that with glutaraldehyde the peptide is bound to the carrier through the NH2-terminal and with BDB through the COOH-terminal handle . Striking effects of the orientation of a synthetic peptide on the immunogenicity and on the specificity of the induced antibodies have been reported (14).…”

Molecular mimicry and myasthenia gravis. An autoantigenic site of the acetylcholine receptor alpha-subunit that has biologic activity and reacts immunochemically with herpes simplex virus.

“…Specific antibodies were eluted using 0.1 M citrate buffer at pH 1.0 and rapidly neutralized with 1 M Tris to pH 7.2. Collections were monitored for protein content at 280 nm (Pharmacia Fine Chemicals), and specificity was confirmed with a monospecific rabbit serum to human IgG (13,16). In preliminary studies, tyrosine was added to the carboxyl terminus labeled with 1251I in order to monitor the amount of peptide bound to thiopropylSepharose.…”

“…All peptides used in this study eluted from the column in a single peak containing at least 80% of the total. For immunization, we synthesized peptides with an additional cysteine as linker and two glycines as spacers at either the amino or carboxy terminus (13). The peptides were coupled at the cysteine to the carrier molecule, keyhole limpet hemocyanin (KLH) by using M-maleimidobenzoyl-N-hydroxysuccinimide ester as cross-linker (13,14).…”

“…For immunization, we synthesized peptides with an additional cysteine as linker and two glycines as spacers at either the amino or carboxy terminus (13). The peptides were coupled at the cysteine to the carrier molecule, keyhole limpet hemocyanin (KLH) by using M-maleimidobenzoyl-N-hydroxysuccinimide ester as cross-linker (13,14). Groups of three New Zealand white rabbits were prebled and immunized sub-cutaneously with 250 ,g of peptide-KLH mixed with complete Freund's adjuvant.…”

Detection of in situ cleaved p115 with the cut specific antibodies in rapid protein inactivation system by tobacco etch viral protease cleavage

“…[12][13][14] Since the cleaved TEV (ENLYFQ) was a hexapeptide, a non-antigenic Ahx was used as a spacer ( Figure 1A) to maximize the presentation of the antigen peptide. The titers were determined by ELISA (not shown).…”