Multiplexed magnetic microsphere immunoassays for detection of pathogens in foods

Abstract

Foodstuffs have traditionally been challenging matrices for conducting immunoassays. Proteins, carbohydrates, and other macromolecules present in food matrices may interfere with both immunoassays and PCR-based tests, and removal of particulate matter may also prove challenging prior to analyses. This has been found true when testing for bacterial contamination of foods using the standard polystyrene microspheres utilized with Luminex flow cytometers. Luminex MagPlex microspheres are encoded with the same dyes as standard xMAP microspheres, but have superparamagnetic properties to aid in preparation of samples in complex matrices. In this work, we present results demonstrating use of MagPlex for sample preparation and identification of bacteria and a toxin spiked into a variety of food samples. Fluorescence-coded MagPlex microsphere sets coated with antibodies for Salmonella, Campylobacter, Escherichia coli, Listeria, and staphylococcal enterotoxin B (SEB) were used to capture these bacteria and toxin from spiked foodstuffs and then evaluated by the Luminex system in a multiplex format; spiked foods included apple juice, green pepper, tomato, ground beef, alfalfa sprouts, milk, lettuce, spinach, and chicken washes. Although MagPlex microspheres facilitated recovery of the microspheres and targets from the complex matrices, assay sensitivity was sometimes inhibited by up to one to three orders of magnitude; for example the detection limits E. coli spiked into apple juice or milk increased 100-fold, from 1000 to 100,000 cfu/mL. Thus, while the magnetic and fluorescent properties of the Luminex MagPlex microspheres allow for rapid, multiplexed testing for bacterial contamination in typically problematic food matrices, our data demonstrate that achieving desired limits of detection is still a challenge.

Fig. 1

A schematic representation of the protocol used in preparing sandwich assays in MagPlex beads for detecting pathogens in food matrices

Fig. 2

Dose–response curves in buffer using MagPlex microspheres for a E. coli, b Salmonella, c Campylobacter, d Listeria, e SEB, and f all bacteria and SEB. Bacteria concentrations are reported in colony forming units (CFU) per milliliter and SEB in nanograms per milliliters. The dashed lines show the fluorescence threshold used to calculate the LODs for each target (lowest tested concentration that has a mean fluorescence signal greater the mean background fluorescence signal plus three times the standard deviation). Microsphere sets are coated with antibodies to the antigens indicated using the following code: E. coli (square), Salmonella (diamond), Campylobacter (triangle), Listeria (circle), SEB (cross)

Fig. 3

Dose–response curves of MagPlex microspheres detecting E. coli in multiplexed assays in matrices: a PBST, b spinach, c chicken wash, and d milk. Concentrations are reported in colony forming units per milliliter. Microsphere sets are coated with antibodies to the antigens indicated using the following code: E. coli (square), Salmonella (diamond), Campylobacter (triangle), Listeria (circle), SEB (cross)

Fig. 4

Dose–response curves of MagPlex microspheres detecting Listeria in multiplexed assays in matrices: a PBST, b spinach, c chicken wash, and d milk. Concentrations are reported in colony forming units per milliliter. Microsphere sets are coated with antibodies to the antigens indicated using the following code: E. coli (square), Salmonella (diamond), Campylobacter (triangle), Listeria (circle), SEB (cross)

Fig. 5

Dose–response curves of MagPlex microspheres detecting Campylobacter in multiplexed assays in matrices: a PBST, b spinach, c chicken wash, and d milk. Concentrations are reported in colony forming units per milliliter. Microsphere sets are coated with antibodies to the antigens indicated using the following code: E. coli (square), Salmonella (diamond), Campylobacter (triangle), Listeria (circle), SEB (cross)

Fig. 6

Dose–response curves of MagPlex microspheres detecting Salmonella in multiplexed assays in matrices: a PBST, b spinach, c chicken wash, and d milk. Concentrations are reported in colony forming units per milliliter. Microsphere sets are coated with antibodies to the antigens indicated using the following code: E. coli (square), Salmonella (diamond), Campylobacter (triangle), Listeria (circle), SEB (cross)

Fig. 7

Dose–response curves of MagPlex microspheres detecting SEB in multiplexed assays in matrices: a PBST, b spinach, c chicken wash, and d milk. Concentrations are reported in colony forming units per milliliter. Microsphere sets are coated with antibodies to the antigens indicated using the following code: E. coli (square), Salmonella (diamond), Campylobacter (triangle), Listeria (circle), SEB (cross)