Comparison of enterobacterial common antigen from different species by serological techniques

Enterobacterial common antigen (ECA) was isolated from a number of selected species (including Salmonella montevideo, Shigella sonnei and Plesiomonas shigelloides) using the extraction method described by Männel and Mayer [Eur. J. Biochem. 86, 361-370 (1978)]. ECA of all these species behaved identically in enzyme-linked immunosorption assay (ELISA) and in its inhibition using monoclonal anti-ECA antibodies. Immunoblotting showed a ladder-like pattern of at least 20 bands for all preparations tested. ECA modified at its lipid moiety (e.g. by phospholipases A2 and D or by mild acid hydrolysis) lost its coating capacity leaving, however, the serological reactivity as detected by inhibition assays intact. In contrast, reduction of the carboxylic groups of 2-acetamido-2-deoxy-D-mannopyranosyluronic acid destroyed the serological reactivity. Deacylated ECA was also not detectable in immunoblotting. Chemical reacylation restored the reactivity of deacylated ECA in ELISA and in immunoblot and thus proved the essential function of fatty acids for the physicochemical properties of the molecule. 2-Acetamido-2-deoxy-D-glucopyranose was identified as the reducing end of the ECA sugar chain after splitting off the lipid moiety by phospholipase D.