Development of a Non-O157:H7 Enterohemorrhagic Escherichia coli (EHEC) Lateral Flow Device

Cesar Nadala, Jason Cantera and Mansour Samadpour

Abstract

A lateral flow device was developed for detection of non-O157:H7 EHEC using polyclonal antibodies generated in goats against each of the top six non-O157:H7 EHEC targets. The device was tested against a panel of bacterial cells as well as on spiked enrichments. It was able to correctly identify and distinguish each target strain from the other five, as well as from other bacteria. It was also able to detect all six strains in 18-hour meat enrichments spiked with 3-6 CFU/375 g of meat.

Introduction

Six strains of non-O157:H7 enterohemorrhagic Escherichia coli (EHEC) have been identified as a serious threat to food safety. In order to address the risk posed by these bacteria, antibodybased rapid tests are needed to complement nucleic acid-based tests that detect and identify these virulent strains. Immunochromatographic assays (also called lateral flow devices) are commonly used to detect a variety of foodborne pathogens including E. coli O157:H7, Salmonella and Listeria. These assays are used in conjunction with nucleic acid based tests to detect and identify the pathogens. We describe here the development of an antibody-based lateral flow device to detect six of the most common non-O157:H7 EHEC strains: O26, O45, O103, O111, O121, and O145.

Methods

Polyclonal antibodies to each of the six EHEC strains (O26, O45, O103, O111, O121, and O145) were produced in goats by injecting the animals with heat-killed bacterial cells. Goat IgGs were affinity-purified using a protein G column and evaluated for purity and activity by SDS-PAGE and ELISA, respectively. The purified goat IgGs were used as both capture and detection reagents in the lateral flow device. The capture reagents were applied onto mylar-backed nitrocellulose membranes using the IsoFlow Dispenser (Imagene Technology, Hanover, NH), overcoated with additive reagent mix (ARM, phosphate buffered saline [pH 7.2], detergent, bovine serum albumin, and sucrose), dried at 45°C and stored desiccated until needed. To prepare the detection reagent, nanogold particles were coated with the goat IgGs specific for each EHEC strain. The gold conjugates were then diluted in ARM and sprayed on to glass fiber conjugate pads using the IsoFlow Dispenser. The pads were then dried and stored desiccated until needed. The lateral flow device consisting of a plastic backing card, nitrocellulose membranes, glass fiber conjugate pads, glass fiber sample pads, printed plastic films, and cellulose absorbent pads was assembled and then cut into strips using a Kinematic Matrix 2360 Programmable Shear (Sonora, CA). See Figure 1 for illustration of the EHEC lateral flow device.

The EHEC lateral flow device is packed in foil bags with desiccant. To perform the test, a strip is taken out of the foil bag and placed on a flat, dry surface. The sample ID is written on the absorbent pad using a pencil. A 200 µl aliquot of the sample (i.e. enrichment medium) is applied to the sample pad. The result is read after 25 minutes. The appearance of the Procedural Control (PC) lines indicates that the test is working properly. The appearance of a clearly visible signal in the Test lines indicates that the corresponding EHEC strain was detected. The absence of a clearly visible signal in the Test lines indicates that none of the six EHEC strains was detected.

The device was initially tested against a panel of purified bacterial cells and then later on spiked meat enrichment samples. Meat samples were spiked at 3-6 CFU/375 g of meat (Table 2). The lateral flow device was tested against real-time PCR (using the the ISO, FSIS, and IEH methods) and multiplex PCR, plus cultural and molecular confirmation (FSIS MLG 5B.00).

EHEC lateral flow device

Figure 1: Illustration of the EHEC lateral flow device.

Goat Plasma graph

Figure 2: Example of goat plasma titration by ELISA

Figure 3: SDS-PAGE of purified antibodies

Figure 3: SDS-PAGE of purified antibodies

 

Table 1: Activity of purified antibodies by ELISA

Table 1: Activity of purified antibodies by ELISA

figure4

Figure 4: Principle of the EHEC lateral flow device: Sample is added in the middle (A), solubilizing the detection reagents and allowing it to bind to the target analyte (*) that in turn is captured in the corresponding test line (B). Procedural control (PC) line captures detection reagent via anti-goat antibodies.

figure5

Figure 5: Testing of different EHEC serotypes on the lateral flow device.

EHEC strains used

 

Table 3: Performance of the EHEC lateral flow device as compared to real-time & standard PCR assays after 18 h incubation at 42°C.

Table 3: Performance of the EHEC lateral flow device as compared to real-time & standard PCR assays after 18 h incubation at 42°C.

Results

High titer antibodies (>1:100,000) to each of the six EHEC serotypes were produced in goats after at least five injections of heat-killed cells (example for E. coli O26, Figure 2). Purified IgG showed greater than 95% purity by SDS-PAGE and Coomassie staining (example for E. coli O45, O111, and O121, Figure 3). The prototype EHEC lateral flow device detected each of the six EHEC strains when tested at 105-106 CFU/ml (Figure 5). The device detected five of the six strains in 12-hour meat enrichments (data not shown) and all six strains in 18-hour meat enrichments spiked with 3-6 CFU/375 g of meat (Table 3).

Discussion

The EHEC lateral flow device is a rapid immunoassay in a bilateral flow test strip format. It detects the presence of six different EHEC strains directly from the enrichment medium sample.

The EHEC lateral flow device is a 4 mm x 100 mm test strip consisting of a sample pad, two conjugate pads containing dried detection reagents, two nitrocellulose membranes lined with Test and Procedural Control lines, and two absorbent pads.
The sample is applied to the sample pad in the middle of the strip and flows in two directions through the test strip by capillary action (Figure 4). As the sample moves through the conjugate pads, it solubilizes the detection reagents consisting of nanogold particles coated with anti-EHEC antibodies. If the EHEC organism is present in the sample, a complex of the organism and nanogold particles will form and be captured by specific anti-EHEC antibodies present in the Test lines, as the complex flows through the nitrocellulose membranes.

Accumulation of this complex in positive samples is indicated by a clearly visible colored signal in the corresponding Test line, while negative samples are indicated by absence of a clearly visible signal in the Test lines. Each test strip has a Procedural Control line downstream of three Test lines at each end of the strip to control for reagent stability and functionality. A clearly visible Procedural Control line indicates that the reagents have flowed normally through the Test strip. The test can be completed in 30 minutes.

Lateral flow device performance is heavily dependent on the specificity and affinity of antibodies used as capture and detection reagents. The fact that it can distinguish between the different serotypes of EHEC indicate that the high titer polyclonal antibodies were directed largely at the O antigen.
The results of the spiked meat testing showed the potential for the EHEC lateral flow device as a tool to be used in conjunction with nucleic acid-based tests to detect and identify six of the most common EHEC serotypes.

Conclusions/Recommendations

  • The EHEC lateral flow device was able to correctly identify and distinguish each target strain from the other five, as well as from other bacteria. It was also able to detect all six strains in 18-hour meat enrichments spiked with 3-6 CFU/375 g of meat.
  • The EHEC lateral flow device can be a valuable tool in efforts to simultaneously screen and detect six strains of non-O157:H7 enterohemorrhagic Escherichia coli (EHEC) from food samples.