Development of ganglioside-based assays for the identification of botulinum and cholera toxins utilizing an evanescent wave biosensor.

摘要

An evanescent wave fiber-optic biosensor was used in an effort to develop an assay for the rapid detection of two biological toxins: cholera toxin and botulinum toxin. The Analyte 2000 fiber-optic biosensor utilizes a sandwich immunoassay format. Gangliosides or liposomes are directly adsorbed to the surface of the fiber-optic waveguide through hydrophobic interactions. The waveguide is exposed to a sample containing the toxin of interest, then subsequently exposed to a polyclonal detection antibody conjugated to the fluorophore cyanine 5. Excitation light from a 635nm laser diode is propagated through the waveguide and fluorescent molecules within approximately 100nm of the waveguide are excited. The emission light from the excited cyanine 5 molecules reverberates into the waveguide and is quantitated in pico Amperes and displayed on a computer.; The exotoxins of Vibrio cholerae and Clostridium botulinum, cholera and botulinum toxin, respectively, were used for potential assay development. Assay development utilizing the biosensor was attempted for the detection of botulinum toxin in buffer. The limit of detection remained too high to generate a positive signal for the detection of botulinum toxin. Biosensor assays were developed to detect cholera toxin in buffer, oyster homogenate, pure culture and induction media.; A cholera toxin standard curve was generated with a limit of detection of 1 ng/ml. The values were normalized by setting 100 ng/ml of cholera toxin to a value of 100. Signals were detected in oyster homogenate spiked at 5 mug/ml as well as unspiked oyster homogenate. A Western blot showed that there were cross reactive proteins in the oyster matrix at molecular weights different from those of the cholera toxin. Cholera toxin production by three strains of Vibrio cholerae with values estimated to range from 100 pg--100 ng was detected with the biosensor. Additionally, oysters were harvested from Tampa Bay and placed in a 10 gallon tank filled with different types of induction media. The tank was inoculated with Vibrio cholerae and the oysters and induction medium were analyzed at varying times for the presence of cholera toxin. Vibrio cholerae cells were viable through 24 hours but no toxin was detectable.