Development of a Highly Sensitive Bead‐ELISA to Detect Bacterial Protein Toxins

Abstract

A highly sensitive sandwich enzyme-linked immunosorbent assay to detect bacterial toxins was developed. Fab' of anti-toxin IgG was conjugated with horseradish peroxidase by the maleimide method and tetramethylbenzidine was used as substrate. As the solid phase, a 6.5 mm diameter polystyrene bead was used and this was coated with the anti-toxin IgG. The entire assay could be completed within 3.5 hr. The sensitivity of this bead-ELISA was found to be quite high with various bacterial toxins : less than 20 pg/ml for thermostable direct hemolysin of Vibrio parahaemolyticus, less than 60 pg/ml for Shiga toxin, less than 20 pg/ml for VT2 (Shiga-like toxin II) of Escherichia coli, less than 200 pg/ml for heat-labile enterotoxin of E. coli, and less than 6 pg/ml for cholera enterotoxin.

High prevalence of thermostable direct hemolysin (TDH)-like toxin inVibrio mimicusstrains isolated from diarrhoeal patients

“…Finally, the reaction was stopped by adding 0-2 ml of 4N H2S04 and the intensity of the resulting yellow colour was measured at 450 nm using a spectrophotometer. For TDH-like toxin, culture filtrate was assayed by using the beads ELISA method (Nissui Pharmaceutical Co.) reported previously [12]. The procedures for ELISA was the same as the beads method for CT detection, except that the polystyrene beads were coated with anti-TDH IgG.…”

In Vitro Inhibition of Cholera Toxin Production in <i>Vibrio cholerae</i> by Methanol Extract of Sweet Fennel Seeds and Its Components

“…The diluted culture was spread onto LB-agar (Difco) plates to measure the CFU/ml in each sample. Quantification of CT production in V. cholerae strains, either in the presence or absence of sweet fennel methanol extract, trans-anethole, or 4-allylanisole, was carried out by bead-ELISA as described previously (20). After 8 h of incubation, the remaining culture was used for preparation of cell free supernatant (CFS).…”

“…Thus, we hypothesized that there may be some spices that can be used for medicinal purposes to treat diarrheal dis- 2) : Quantification of CT production (ng/ml) in V. cholerae strains without sweet fennel (SF) seeds methanol extract measured by bead-ELISA as described in Oku et al (20). Data are presented as the average ± SD of 3 test samples (ng/ml).…”

Genetic and Immunological Analysis of a Novel Variant of Shiga Toxin 1 from Bovine Escherichia coli Strains and Development of Bead‐ELISA to Detect the Variant Toxin

“…Polyclonal rabbit antisera against either anti-Stx1v52 or Stx1 was prepared as described previously (44). Preparation of conjugates by conjugating Fab' of antitoxin IgG with horseradish peroxidase by the maleimide method was as described previously (28). To make the Stx1v52 conjugate, Fab' of Stx1v52 conjugate prepared as above was applied to an Stx1-immobilized (Stx1-CNBr activated) Sepharose 4B column (1.0ϫ7.0 cm) and anti-Stx1v52 antibody in common with anti-Stx1 was absorbed.…”

“…As the solid phase, polystyrene beads coated with the anti-toxin IgG was used as described previously (28).…”