Neutralization receptor-based immunoassay for detection of neutralizing antibodies to Escherichia coli verocytotoxin 1

Abstract

The NeutRELISA, a modification of the receptor enzyme-linked immunosorbent assay developed for the detection of verocytotoxin 1 (VT1) which permits the rapid detection of neutralizing antibodies (NAb) against this toxin, has been performed. A standard concentration of VT1 was preincubated with VT-immune or nonimmune rabbit serum. The serum-toxin mixtures were then added to microtiter plates coated with deacylated globotriosyl ceramide (lyso-Gb3). The reduction of VT1 binding to lyso-Gb3 in the immune serum-toxin mixtures compared with the VT1-Gb3 binding in the nonimmune serum-toxin mixtures was detected by using mouse monoclonal antibody to VT1. After standardization of the NeutRELISA with rabbit sera, 57 human control serum samples were tested to establish a cutoff value below which NeutRELISA results would be considered positive. Thirty-three single serum samples known to demonstrate NAb to VT1 by biological assay reproducibly demonstrated VT1 NAb when tested by the NeutRELISA. There was a close correlation between the biological VT1 neutralization assay and the NeutRELISA. This assay offers a practical, rapid, and reliable approach for the detection of NAb to VT1 and other verocytotoxins.