Cross-reactivity of monoclonal antibodies to Escherichia coli J5 with heterologous gram-negative bacteria and extracted lipopolysaccharides.

Abstract

A panel of eight murine monoclonal antibodies (MAbs) were produced against heat-killed Escherichia coli J5 and shown to react with J5 lipopolysaccharide (LPS) by enzyme-linked immunosorbent assay (ELISA). These antibodies were then assayed by a suspension ELISA for reactivity with up to 20 heterologous smooth or rough isolates of gram-negative bacteria, which were assayed after heat or formalin treatment, or as live cells. Extracted LPS from the same bacteria were tested for reactivity with the MAbs by direct ELISA. The MAbs demonstrated broad cross-reactivity with most heat-treated bacteria. In contrast, cross-reactivity of the MAbs with live or formalin-treated bacteria was limited almost exclusively to E. coli J5, Hemophilus species, or rough mutants of Salmonella minnesota. Reactivity with extracted LPSs and lipid A varied considerably depending on the MAb. Further, when Western blotting was used as the assay only four of eight MAbs reacted with J5 LPS, and none of the MAbs reacted with LPS from smooth S. minnesota or any of its rough mutants. Adsorption of the MAbs with acid hydrolyzed, boiled, or live E. coli J5 prior to ELISA of the MAbs with J5 LPS supported evidence that none of the MAbs were specific for lipid A and that reactivity was greater with boiled than with live cells. Thus, the cross-reactivity of antibodies to E. coli J5 LPS is dependent on the physical state of the bacteria or LPS used for assay, the assay used, and the specificity of the antibody.