Enterobacterial common antigen (ECA) is a family‐specific surface antigen shared by all members of the Enterobacteriaceae and is restricted to this family. It is found in freshly isolated wild‐type strains as well as in laboratory strains like Escherichia coli K‐12. The family specificity of ECA can be used for taxonomic and diagnostic purposes. ECA is located in the outer leaflet of the outer membrane. It is a glycophospholipid built up by an aminosugar heteropolymer linked to an l‐glycerophosphatidyl residue. In a few rough mutants, in addition, the sugar chain can be bound to the complete lipopolysaccharide (LPS) core. Recently, for Shigella sonnei a lipid‐free cyclic form of ECA was reported. The genetical determination of ECA is closely related to that of lipopolysaccharide. For biosynthesis of ECA and LPS partly the same sugar precursors and the same carrier lipid is used.

No abstract

A series of monoclonal antibodies directed against lipid A was characterized by using synthetic lipid A analogs and partial structures. These compounds vary in phosphate substitution, acylation pattern (type, number, and distribution of fatty acids), and, in the case of monosaccharides, in their backbone glycosyl residue. The monoclonal antibodies tested could be subdivided into five groups according to their reactivity patterns. One group reacted exclusively with 1,4'-bisphosphoryl lipid A, and a second also reacted with 4'-monophosphoryl lipid A. Two further groups recognized either 4-phosphoryl or 1-phosphoryl monosaccharide partial structures of lipid A. The fifth group reacted with 4-phosphoryl monosaccharide structures and with phosphate-free compounds. Antibodies reactive with monosaccharide structures also recognized their epitopes in corresponding phosphorylated disaccharide compounds. Both groups of monosaccharide and monophosphoryl lipid A-recognizing antibodies have access to their epitopes in bisphosphoryl compounds as well. Because of this unidirectional reactivity with more complex structures, the various specificities cannot be distinguished by using bisphosphoryl lipid A (e.g., Escherichia coli lipid A) as a test antigen. The epitopes recognized by the various monoclonal antibodies all reside in the hydrophilic backbone of lipid A, and there was no indication that fatty acids were part of the epitopes recognized. Nevertheless, the reactivities of compounds in the different test systems are strongly influenced by their acylation patterns; i.e., acyl groups may modulate the exposure of lipid A epitopes. * Corresponding author. ical assay system used. Thus, in the present study, passive immunohemolysis and enzyme immunoassay (EIA) were compared. We will show that the specificities characterized in polyclonal sera are indeed also expressed by MAbs, as estimated in all serological assay systems employed. MATERIALS AND METHODS Bacterial lipid A. Bisphosphoryl lipid A (lipA-Ac) and 4'-monophosphoryl lipid A (lipA-HCl) were obtained from Escherichia coli Re mutant F515 by treating phenol-chloroform-petroleum ether-extracted LPS with either acetate buffer or HCl (5). Synthetic lipid A and partial structures. The structures of synthetic disaccharide antigens and the various substituents are shown in Fig. 1 and Table 1. Compounds LA-15-PP, LA-15-PH, LA-15-HP, and LA-15-HH represent synthetic hexaacyl E. coli lipid A and its 4'-phosphoryl, 1-phosphoryl, and 1,4'-dephosphoryl partial structures, respectively (23, 29). Preparations LA-14-PP, LA-14-PH, LA-14-HP, and LA-14-HH (24) correspond to tetraacyl precursor Ia (22) and its dephosphoryl derivatives. Compounds LA-19-PP, LA-19-PH, and LA-19-HP represent synthetic counterparts of alkali-treated (de-O-acylated) lipid A and its monophosphoryl derivatives. LA-16-PP corresponds to the heptaacyl species of Salmonella minnesota lipid A (17). LA-20-PP represents synthetic pentaacyl precursor lb (22, 40), and LA-20-PH represents its 4'-monophosphoryl partial structur...

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 Mannel and Mayer [Eur. J. Biochem. 86, 361-370 (1978)l. 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-~-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-~-glucopyranose was identified as the reducing end of the ECA sugar chain after splitting off the lipid moiety by phospholipase D.