Rapid Simultaneous Ultrasensitive Immunodetection of Five Bacterial Toxins

Abstract

Rapid ultrasensitive detection of gastrointestinal pathogens presents a great interest for medical diagnostics and epidemiologic services. Though conventional immunochemical and polymerase chain reaction (PCR)-based methods are sensitive enough for many applications, they usually require several hours for assay, whereas as sensitive but more rapid methods are needed in many practical cases. Here, we report a new microarray-based analytical technique for simultaneous detection of five bacterial toxins: the cholera toxin, the E. coli heat-labile toxin, and three S. aureus toxins (the enterotoxins A and B and the toxic shock syndrome toxin). The assay involves three major steps: electrophoretic collection of toxins on an antibody microarray, labeling of captured antigens with secondary biotinylated antibodies, and detection of biotin labels by scanning the microarray surface with streptavidin-coated magnetic beads in a shear-flow. All the stages are performed in a single flow cell allowing application of electric and magnetic fields as well as optical detection of microarray-bound beads. Replacement of diffusion with a forced transport at all the recognition steps allows one to dramatically decrease both the limit of detection (LOD) and the assay time. We demonstrate here that application of this "active" assay technique to the detection of bacterial toxins in water samples from natural sources and in food samples (milk and meat extracts) allowed one to perform the assay in less than 10 min and to decrease the LOD to 0.1-1 pg/mL for water and to 1 pg/mL for food samples.

Magnetic Particle-Scanning for Ultrasensitive Immunodetection On-Chip

“…As seen from the curves shown in ref. 43,44 , the 1/ value is quite low in this type of devices (below 10 10 Ag). The plateau, on the right side of the sigmoid, can be observed on the same range of concentrations considered in our device, indicating concentrations above the 1/ limit.…”

“…Interestingly, for this type of device, the concentration response drawn over a logarithmic scale typically is a sigmoid saturating at high concentrations. 43,44 We note that a sigmoid is a good approximation of a Langmuir isotherm on a logarithmic scale. The flattening of the curve typically limits the capabilities of these chips at very high concentrations.…”

Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

“…To permit the sensitive observation of the antigen-antibody reaction, antigens or antibodies have to be labeled either directly or indirectly. Protocols for indirect labeling include functionalized secondary antibodies and the biotin-avidin system to bridge the antigen-antibody reaction and signal generation (Figure 2D) [23,24]. Direct modification of the primary antibody can be achieved by biomolecules, such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP), and may result in decreased affinity and stability induced by unspecific side effects of the coupling chemistry and/or steric hindrance by the attachment of the reporter enzymes.…”

“…Copyright 2013 American Chemical Society; ( B ) Scheme of streptavidin functionalized magnetic nanoparticles (MNPs) for the simultaneous detection of five bacterial toxins in a microarray. Reprinted with permission from [24]. Copyright 2013 American Chemical Society; ( C ) Scheme of indirect competitive immunoassay for the detection of microcystin-LR based on the conjugation of quantum dots (QDs) and aminoethyl microcystin-LR.…”

Comparison of DOT-ELISA and Standard-ELISA for Detection of the Vibrio cholerae Toxin in Culture Supernatants of Bacteria Isolated from Human and Environmental Samples

“…Since clinical cases of severe watery diarrhoea are found to be associated with the isolation of toxin producing bacteria, the identification of cholera toxin is an important step in the diagnosis of cholera disease. Indeed, the detection of cholera toxin has been evaluated using animals models [2], cell lines, immunoassays and molecular-based assays [3][4][5]; most of these techniques are time-consuming and the mRNA synthesis not always correlates with protein expression. Because the only way to confirm the presence of the cholera toxin is by mean of laboratory diagnosis, the aim of this work was to compare DOT-ELISA and Standard-ELISA for detection of the Vibrio cholerae toxin in supernatants of bacteria isolated from human and environmental samples.…”