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 ...
Vol. 28, No. 12
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1990, p. 2830-2833 0095-1137/90/122830-04$02.00/0 Copyright © 1990, American Society for Microbiology
Neutralization Receptor-Based Immunoassay for Detection of Neutralizing Antibodies to Escherichia coli Verocytotoxin 1 J. BOULANGER,' M. PETRIC,"2 C. LINGWOOD,12 34'5 H. LAW,3 M. ROSCOE,' AND M. KARMALI12* Departments of Microbiologyl* and Biochemistry? Hospital for Sick Children, 555 University Avenue, Toronto, Ontario MSG IX8, and Departments of Microbiology,2 Biochemistry,4 and Clinical Biochemistry,5 University of Toronto, Toronto, Ontario M5F IA8, Canada Received 2 July 1990/Accepted 7 September 1990
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.
assay were selected for testing by the NeutRELISA. Positive sera encompassed a range of VT1 NAb titers from 1:4 to 1:128. VT1. VT1 was purified (7) from recombinant E. coli JB28 (kindly provided by J. Brunton). The protein content of the VT1 was 0.5 mg/ml, and the cytotoxicity titer was 107. Mouse monoclonal antibody to VT1. We produced a mouse monoclonal neutralizing antibody (MAb) to VT1 (PH1) by a standard technique (9) using repeated sublethal doses of VT1 for immunization (9). The specificity of MAb PH1 for the VT1 B subunit was confirmed by Western blotting (immunoblotting), and the antibody showed no cross neutralization of VT2, VTE, or Shiga-like toxin II (C. Lingwood, unpublished data). Glycolipids. Gb3 was purified from human kidney and deacylated as previously described (1). Neutralization assay with Vero cells. All antisera (human test sera and rabbit sera used in the development of the assay) were tested for VT1 NAb as previously described (3,
Infection with verocytotoxin (VT)-producing Escherichia coli (VTEC) is associated with a spectrum of diseases, including diarrhea, hemorrhagic colitis, and the hemolytic uremic syndrome (HUS). Early studies strongly suggested a causal link between these diseases and VTEC infection on the basis of the detection of VTEC and neutralizable free fecal VT in stools and of rising titers of neutralizing antibodies (NAb) to VT in persons suffering from these illnesses (3-6, 8). The standard methods for determination of free fecal VT and NAb to VT involve cytotoxicity and cytotoxin neutralization assays which use Vero cells in culture. These assays are slow, labor-intensive, and difficult to standardize, and they have hampered studies of the epidemiology of VTEC infection. A rapid, easily performed, sensitive, and specific receptorspecific enzyme-linked immunosorbent assay (RELISA) for the detection of Escherichia coli VT has recently been described (1). The assay is based on the affinity of VT1 for the glycolipid globotriosyl ceramide (Gb3) that is de-Nacylated (lyso-Gb3). The extreme sensitivity of this assay made it possible to detect very low levels of VT1. In this paper we describe a modification of the RELISA for the detection of NAb to VT1 (NeutRELISA). Sera. Rabbit anti-VT antibodies against purified VT1 toxoid were available from stock previously produced in our laboratory (7). The immune rabbit sera had a VT1 NAb titer of 1:4,096 and the preimmune rabbit sera had a VT1 NAb titer of
