Enterobacterial common antigen: Isolation from Shigella sonnei, purification and immunochemical characterization [rabbit, coat erythrocytes]

In the studies presented the effective procedure of isolation and purification of enterobacterial common antigen from Shigella sonnei has been elaborated. The method is based on sonification of bacterial suspension in the presence of lysozyme and EDTA and subsequent extraction of the pellet with boiling water. The crude extract of common antigen was purified by fractionation with ethanol and chromatography on silica gel and Sephadex LH‐20. The comparison of several extraction procedures of enterobacterial common antigen from Shigella sonnei proved that the method described above is most effective. The purified enterobacterial common antigen preparation obtained preserved full biological activity: antigenicity (precipitation and activity in enzyme‐linked immunosorbent assay), immunogenicity in rabbits, ability to coat erythrocytes (passive hemagglutination) and inhibitory activity in passive hemagglutination. The pure enterobacterial common antigen was identified to 90% as a polymer of N‐acetyl‐D‐mannosaminuronic acid and N‐acetyl‐D‐glucosamine (2:1, molar ratio), O‐acetylated and containing 3.2% fatty acids (C16:0 and C18:1, not oleic). It contains 5.3% nitrogen, > 4% protein, ≥ 0.5% phosphorus and ≥ 1.6% neutral sugar; glycerol and RNA were not found in the preparation.