SF6034 - Fluorescence Polarization Immunoassays (FPIA) for Food Safety: A Rapid Detection System for Pathogens and Chemicals

Researcher:

Dr. J. Christopher Hall, Dept. of Environmental Biology, University of Guelph

Objectives:

• To expand the utility of food/water-borne pathogen/chemical-specific antibodies for rapid, accurate and simple use in both laboratory and field settings by formatting them as fluorescence polarization immunoassays (FPIAs).

Expected Benefits:

• To develop and validate a proven antibody-based technology, Fluorescence Polarization Immunoassay (FPIA), to the area of rapid food and water safety diagnostics.

Summary of Research Results:

FPIA is based on the rotation rate of molecules in solution, where smaller molecules rotate more quickly than larger molecules. When a smaller molecule is bound to a larger molecule, such as an antibody, the rotation rate is subsequently decreased, and this decrease is measurable if the smaller molecule is labeled with a fluorescent tag. Several FPIA formats were studied using agrochemicals, toxins and a food pathogen to determine whether this technology was quicker to perform yet equivalent in sensitivity to ELISA technology.

The sensitivity of FPIA was similar to ELISA when detection of the fungicide azoxystrobin, West Nile virus control chemicals diflubenzuron and novaluron (i.e. control of virus through mosquito control), and of the mycotoxin contaminant of cereal products known as DON toxin were assayed. FPIA, however, was the least labor-intensive and required the least time to perform. ELISAs required approximately 3 h to perform following incubation overnight with coating conjugate. In contrast, the FPIAs required less than 90 min to perform and no coating of plates was required.

A model for detection of transgenic plant-produced recombinant protein was also investigated to determine if FPIA is capable of discriminating between transgenic and non-transgenic plants. FPIA was capable of discriminating between the transgenic and non-transgenic plants using crude protein extracts from leaves of transgenic and wild-type (non-transgenic) tobacco. This technology could thus be adapted for verification of organically-grown produce, allowing for certification of agricultural products that are free of genetically modified materials, although fluorescent probes for each recombinantly-expressed  protein would need to be developed.
 
FPIA was able to detect Salmonella in contaminated pools of eggs with a sensitivity of 10 colony-forming units (CFU) per ml, but only if a 72-hr enrichment step was included. Amplification of microorganisms (enrichment) is therefore required to reach the government required detection level of 1 CFU per 25 g of food, excluding FPIA as the technology of choice for pathogen detection in food.

FPIA sensitivity levels are within the limits set by the Ontario government (usually PPB to PPM) in cases of small molecule food contaminants such as proteins and pesticides, which cannot be amplified or detected by real-time PCR or DNA microchip assays. Thus the power of FPIA technology likely lies within further assay development for rapid small molecule detection (agrochemicals, proteins and toxins). FPIA technology would be useful at replacing ELISA technology in food safety diagnostics applications as it quicker and easier to perform. FPIAs offer competitive advantages over traditional immunoassay technologies because they are rapid, easier and less expensive to perform, and portable.  FPIAs can generally be performed in seconds to minutes and are amenable to high-throughput screening, where a single instrument can perform up to 50,000 samples per day, as well as for field use, as portable FPIA diagnostic instruments have been developed.

Although FPIAs may be more susceptible than other immunoassay formats to background interference from naturally fluorescing compounds that may exist in sample matrices, the hapten- and pathogen-FPIAs that were developed during the course of this research did not suffer matrix effect interference. Nevertheless, it is strongly recommended that potential matrix effects should be investigated on a case-by-case basis.