Simultaneous direct detection of toxigenic and non-toxigenic Vibrio cholerae from rectal swabs and environmental samples by sandwich ELISA.
A mAb-based simple, specific and rapid two-tip dipstick ELISA was developed for simultaneous detection of toxin- and non-toxin-producing strains of Vibrio cholerae, and for direct detection of V. cholerae from rectal swabs of patients and from environmental water samples. Rabbit polyclonal antibodies and murine mAbs were raised against recombinant protein (r-protein) antigens of cholera toxin B (CtxB) and outer membrane protein W (OmpW). Rabbit polyclonal antibodies to both r-proteins were coated individually onto the tips of nitrocellulose (NC) membranes of a two-tipped NC dipstick as capture antibodies and a mixture of two mAbs was used for the detecting antibodies. The test was found to be specific for V. cholerae strains O1, O139, non-O1 and non-O139, and did not show any cross-reaction to closely related bacterial strains. The test was evaluated on rectal swabs collected at the bedside of 75 hospitalized diarrhoeal patients and on 50 environmental water samples after enrichment for 4 h in alkaline peptone water. The mAb two-tip dipstick ELISA detected V. cholerae in 52/75 rectal swabs and 2/50 environmental water samples for CtxB antigen, and in 1/50 environmental water samples for the non-toxin OmpW antigen of V. cholerae within 1.5 h. These findings were identical to those observed using PCR and conventional culture methods. Thus, this mAb-based two-tip dipstick ELISA could be used for early and reliable simultaneous detection of toxigenic and non-toxigenic strains of V. cholerae from clinical and environmental water samples.
Tuteja
Urmil
U
Division of Microbiology, Defence Research and Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, India.
Kumar
Sanjay
S
Division of Microbiology, Defence Research and Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, India.
Shukla
Jyoti
J
Division of Microbiology, Defence Research and Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, India.
Kingston
Joseph
J
Division of Microbiology, Defence Research and Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, India.
Batra
Harsh V
HV
Division of Microbiology, Defence Food Research Laboratory, Siddharth Nagar, Mysore, Karnataka, India.
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000032:analysis*; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology; D002772:Cholera Toxin / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005243:Feces / Q000737:chemistry* / Q000382:microbiology; D006801:Humans; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014734:Vibrio cholerae / Q000737:chemistry* / Q000302:isolation & purification; D014867:Water / Q000737:chemistry*; D014871:Water Microbiology
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D014867:Water; D002772:Cholera Toxin