The Immunochemistry of Peptidoglycan. Antibodies Against a Synthetic Immunogen Cross-reacting with an Interpeptide Bridge of Peptidoglycan

An albumin-peptide conjugate was synthesized, which carries pentaglycine peptides with C-terminal glycine residues as found in the interpeptide bridges of the peptidoglycan of many staphylococci. Immunization of rabbits with this synthetic immunogen yielded antisera containing predominantly antibodies against the peptide moiety of the conjugate. The quantitative precipitin and the Ouchterlony agar gel reaction with several synthetic protein-peptide-conjugates, immunoaffinity chromatography of the antisera on Sepharose-(Gly-Gly-Gly-Gly-Gly)n and hapten inhibition studies with several synthetic peptides and peptide derivatives demonstrated that the antibodies were highly specific for oligoglycine peptides with C-terminal glycine. These antibodies also reacted strongly with staphylococcal peptidoglycans with an interpeptide bridge composed of pentaglycine peptides or of pentaglycine peptides in which part of the glycine residues were replaced by L-serine. In contrast, all the peptidoglycans lacking interpeptide bridges composed of glycine residues gave no precipitin reaction at all. The final proof for identical determinant groups of albumin-(CH2CO-Gly-Gly-Gly-Gly-Gly)31 and the staphylococcal peptidoglycans applied in the precipitin reaction was furnished by double gel diffusion studies and by hapten inhibition of the precipitin reaction between antisera to albumin-(CH2CO-Gly-Gly-Gly-Gly-Gly)31 and the corresponding peptidoglycans. For rapid screening of the different peptidoglycans, a latex agglutination test was elaborated. Purified antibodies were adsorbed to latex particles, and the titers with the particular peptidoglycans were then determined. The test was highly sensitive, in that 10 nanograms of peptidoglycan could still be detected.