Isolation and Chemical Characterization of the Enterobacterial Common Antigen

Abstract

The isolation of the soluble form of the Enterobacteriaceae common antigen from Salmonella montevideo was achieved by a combination of the phenol/water method and the phenol/chloroform/petroleum ether extraction procedure. A phenol‐soluble fraction highly enriched in the antigen was obtained which was further purified by DEAE‐cellulose chromatography. A fraction eluted with 0.9 M ammonium acetate/methanol was highly active in coating erythrocytes for passive hemagglutination with antisera specific for enterobacterial common antigen and was an active inhibitor of the hemagglutinating system for this antigen. Chemical analyses showed enterobacterial common antigen to be a linear polymer of 1,4‐linked N ‐acetyl‐ d ‐glucosamine and N ‐acetyl‐ d ‐mannosaminuronic acid units esterified to a small extent by palmitic and acetic acids. These amino sugar and fatty acid components represent about 65–70% of the material. The presence of additional still unknown lipid components which might account for the missing 30% and for the low solubility of the isolated antigen in water is discussed. The molecular weight of the readily soluble part of the inhomogeneous isolated enterobacterial common antigen was determined by sedimentation studies in an analytical ultracentrifuge using methanol as solvent; it was found to be 2700.