Identification and strain differentiation of Vibrio cholerae by using polyclonal antibodies against outer membrane proteins

Abstract

Cholera is caused only by O1 and O139 Vibrio cholerae strains. For diagnosis, 3 working days are needed for bacterial isolation from human feces and for biochemical characterization. Here we describe the purification of bacterial outer membrane proteins (OMP) from V. cholerae O1 Ogawa, O1 Inaba, and O139 strains, as well as the production of specific antisera and their use for fecal Vibrio antigen detection. Anti-OMP antisera showed very high reactivity and specificity by enzyme-linked immunosorbent assay (ELISA) and dot-ELISA. An inmunodiagnostic assay for V. cholerae detection was developed; this assay avoids preenrichment and costly equipment and can be used for epidemiological surveillance and clinical diagnosis of cases, considering that prompt and specific identification of bacteria is mandatory in cholera.

FIG. 1

Reactivities of polyclonal antibodies prepared with OMP against bacterial antigens from V. cholerae O1 Ogawa (♦), V. cholerae O1 Inaba (●), V. cholerae O139 (▴), and V. alginolyticus (▵) and with Roshka antigens from V. cholerae O1 Ogawa (⋄) and V. cholerae O1 Inaba (○), as well as with the enteric bacteria listed in Materials and Methods (×).

FIG. 2

The specificity of anti-Vibrio antibodies was defined using several CFU values for V. cholerae O1 Ogawa (♦), V. cholerae O1 Inaba (●), V. cholerae O139 (▴), and enteric bacteria listed in Materials and Methods (×). (a) Anti-V. cholerae O1 Ogawa (diluted 1:50,000); (b) anti-V. cholerae O1 Inaba (diluted 1:50,000); (c) anti-V. cholerae O139 (diluted 1:150,000).

FIG. 3

Evaluation of anti-V. cholerae antibodies by dot-ELISA. Bacteria were added to human fecal samples, adsorbed to PVDF membranes, and reacted with anti-V. cholerae O1 Ogawa (1 and 2), anti-V. cholerae Inaba (3 and 4), and anti-V. cholerae O139 (5 and 6). Bacteria (10⁸ CFU/ml) used were V. cholerae O1 Ogawa (1), V. cholerae Inaba (3), V. cholerae O139 (5), and enteric bacteria (2, 4, and 6; listed in Materials and Methods). Antisera were diluted 1:500, 1:1,000, 1:2,000, 1:4,000, 1:8,000, and 1:16,000 from left to right.