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Comparative Study
. 1999 Sep;6(5):701-4.
doi: 10.1128/CDLI.6.5.701-704.1999.

A reverse-sandwich enzyme-linked immunosorbent assay for verocytotoxin 1 and 2 antibodies in human and bovine sera

H Miyazawa  1 , H Bannai, T Yanase, C Morita, S Satoh, J Sugiyama, S Taniguchi, S Inouye
Affiliations
Comparative Study

A reverse-sandwich enzyme-linked immunosorbent assay for verocytotoxin 1 and 2 antibodies in human and bovine sera

H Miyazawa et al. Clin Diagn Lab Immunol. 1999 Sep.

Abstract

A reverse-sandwich enzyme-linked immunosorbent assay (ELISA), in which an antibody is sandwiched by antigens, was established for the titration of antibodies to verocytotoxins (VT) in human and animal sera. This assay has two advantages over a conventional indirect ELISA: (i) higher specificity and sensitivity and (ii) the ability to comparably titrate antibodies from different species. The VT1 (Shiga-like toxin 1) antibody-positive rates were 5% in 202 normal adult humans and 99% in 93 normal cattle at a dairy farm. This ELISA is most suitable for seroepidemiologic studies of infections with VT-producing Escherichia coli in humans and various animal species.

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Figures

FIG. 1
FIG. 1
Different principles for antibody assays by the RS ELISA and the indirect ELISA. Ab, antibody; Ag, antigen.
FIG. 2
FIG. 2
Antibody titration curve for anti-VT1 hyperimmune rabbit serum. Fourfold serial dilutions of the serum were added to microplate wells in the RS ELISA. Antibody activity was expressed as FU.
FIG. 3
FIG. 3
Distribution of VT1 and VT2 antibody activities (AbU/ml) in human sera. (a) Normal adults. (b) VTEC-infected persons.
FIG. 4
FIG. 4
Inhibition of VT1 antibody activity by VT1 antigen in the RS ELISA. Serum was mixed with 1 μg of VT1 or VT2 per ml before the assay. ELISA activity is shown in FU.
FIG. 5
FIG. 5
Comparison of the RS ELISA with the indirect ELISA for the detection of VT1 antibody in 30 human adult sera. The abscissa shows RS ELISA activity (AbU/ml). The ordinate shows indirect ELISA activity as the absorbance (optical density at 410 nm [OD 410]). The cutoff value (mean + two standard deviations) in the indirect ELISA was determined from the absorbance value of 20 RS ELISA-negative sera (○).
FIG. 6
FIG. 6
Distribution of VT1 and VT2 antibody activities (AbU/ml) in normal cattle sera. Sera from 93 cattle at a dairy farm were titrated for VT1 and VT2 antibody activities by the RS ELISA.
FIG. 7
FIG. 7
Comparison of VT antibody (Ab)-positive rates between cattle and humans who were healthy or VTEC infected.
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