Ganglioside-liposome immunoassay for the detection of botulinum toxin.

A rapid and highly sensitive receptor immunoassay for botulinum toxin (BT) has been developed using ganglioside-incorporated liposomes. Botulism outbreaks are relatively rare, but their results can be very severe, usually leading to death from respiratory failure. To exert their toxicity, the biological toxins must first bind to receptors on the cell surface, and the trisialoganglioside GT1b has been identified as the cell receptor for BT. Therefore, in this study, GT1b was used to prepare the ganglioside-liposomes by spontaneous insertion into the phospholipid bilayer. In a sandwich-based, hybrid receptor immunoassay, BT is detected as a colored band on a nitrocellulose membrane strip, where BT bound to the GT1b-liposomes are captured by anti-BT antibodies immobilized in a band across the strip. The intensity of the colored band can be visually estimated, or measured by densitometry using computer software. The limit of detection (LOD) for BT in the lateral-flow assay system was 15 pg mL(-1), which is comparable to the limits of detection achieved with the most sensitive assays previously reported. However, this rapid assay can be completed in less than 20 min. These results demonstrate that the sandwich assay using GT1b-liposomes for detection of BT is rapid and very sensitive, suggesting the possibility for detecting BT in field screening, simply and reliably, without the need for complex instrumentation.

Ganglioside-liposome immunoassay for the ultrasensitive detection of cholera toxin.

An extremely sensitive bioassay has been developed for cholera toxin (CT) detection, using ganglioside-incorporated liposomes. Cholera is a diarrheal disease, often associated with water or seafood contamination. Ganglioside GM1 was used to prepare the liposomes by spontaneous insertion into the phospholipid bilayer. CT recognition and signal generation is based on the strong and specific interaction between GM1 and CT. In a sandwich immunoassay, CT was detected as a colored band on the nitrocellulose membrane strip, where CT bound to GM1-liposomes can be captured by immobilized antibodies. The intensity of the band could be visually estimated or measured by densitometry, using computer software. The limit of detection (LOD) of CT in the assay system was found to be 10 fg/mL which is equivalent to 8 zmol in the 70-microL sample. The assay was also tested with water samples spiked with CT, providing a LOD of 0.1-30 pg/mL, which is much better than previously reported limits of detection from other assays. The assay could be completed within 20 min. These results demonstrate that the bioassay developed for CT is rapid and ultrasensitive, suggesting the possibility for detecting CT, simply and reliably, in field screening.