Detection of Enterohemorrhagic E. Coli (EHEC) In Food Products and Food Ingredients by The Assurance® EHEC Enzyme Immunoassay (EIA) Method

Laboratory Procedure
MFLP-81
June 2006

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Don Warburton
and the Microbiological Methods Committee
Evaluation Division, Bureau of Microbial Hazards,
Food Directorate, PL2204A1
Health Products and Food Branch (HPFB)
Ottawa, Ontario, K1A 0L2

E-mail: Don_Warburton@hc-sc.gc.ca

1. Application

The method is applicable to the detection of enterohemorrhagic E. coli O157 from food products and food ingredients to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act.

This revised method replaces MFLP-81, dated September 1995.

2. Description

Assurance EHEC EIA is an enzyme immunoassay that detects enterohemorrhagic E. coli (EHEC) including E. coli O157:H7. The Assurance EHEC EIA employs highly specific antibodies directed against EHEC antigens and has been specifically formulated to minimize cross-reactivity with many Enterobacteriaceae. The method has been shown to produce satisfactory results with contaminated foods in AOAC (8.1 - 8.3) and HPFB studies (unpublished data). This method can be used successfully for the detection of EHEC in food products, food ingredients, and environmental sAmples.

3. Principle

The Assurance® EIA method uses proprietary antibodies with high specificity to EHEC antigens bound to microwell plates. Appropriately enriched samples and positive controls are added to plates; any EHEC antigens present will bind to the microwells, forming an antibody-antigen complex. Nonreactive sample material is washed away. An alkaline phosphatase antibody conjugate is added, and, after incubation, unbound conjugate is washed away. The substrate, p-nitrophenylphosphate, is added; absorbance of the resulting enzyme product is read at 405-410 nm.

4. Definition of Terms

See Appendix A of Volume 3.

5. Collection of Samples

See Appendix B of Volume 3.

6. Materials and Special Equipment

The media listed below are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 1 for the formulae of individual media.

1) Assurance® EIA for EHEC (BioControl Systems Inc. 12822 SE 32^nd Street, Bellevue, WA, 98005,USA, phone: (425) 603-1123, or (800) 245-0113, FAX: (425) 603-0070)

2) Modified Trypticase Soy Broth and Novobiocin (mTSB-n).

3) Colworth Stomacher 400, blender or equivalent

4) Incubators capable of maintaining 35°-37°C or 42°C (see 7.3.3.)

5) Water bath capable of maintaining 100°C (or flowing steam autoclave set at 100°C)

6) Microplate washer (optional)

7) Microplate reader, photometer with 405-410 nm filter, capable of reading microtiter plates

8) Filter stomacher bags, with mesh inner bag (VWR Scientific) or equivalent.

9) Filter tips for pipettor (FiltaFlex, Almonte, ON, Tel/FAX: (613)256-3066), or equivalent.

10) Triton X-100

7. Procedure

Each sample unit may be analyzed individually or the analytical units may be combined. Carry out the test in accordance with the following instructions:

7.1 Handling of Sample Units

7.1.1 Keep sample units refrigerated (0-5°C) or frozen, depending on the nature of the product. Shelf-stable foods do not need to be refrigerated. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death.

7.1.2 Analyze sample units as soon as possible after their receipt in the laboratory.

7.2 Preparation for Analysis

7.2.1 Have ready sterile prewarmed mTSB-n.

7.2.2 Clean the surface of the working area with a suitable disinfectant.

7.3 Preparation of Sample

7.3.1 To ensure a truly representative analytical unit agitate liquids or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit. To reduce the workload, the analytical units may be combined for analysis. It is recommended that a composite contain not more than 500 g.

7.3.2 Prepare a 1:10 dilution of the food by aseptically blending or stomaching 25 g or 25 mL (the analytical unit) into 225 mL of the prewarmed mTSB-n broth. Some spices have to be analysed using a higher dilution and with K₂ SO₄ (see MFLP-80).

7.3.3 Incubate overnight (at least 18 hrs) at 35-37°C or 42°C.

7.4 Preparation of sample for EIA analysis

7.4.1 After incubation, thoroughly mix the enriched sample (mTSB-n broth). After allowing suitable time for particles to settle out, transfer 1.0 mL into a test tube.

Retain the enriched sample (mTSB-n) at 2 - 8°C for confirmation of presumptive positive results (Section 7.10).

7.4.2 Inactivate microorganisms by submersing the 1.0 mL aliquot in a boiling water bath for 10 minutes. Cool tubes to room temperature before testing. Tubes that have been boiled can be refrigerated @ 4° - 8°C up to four days prior to testing

7.5 Reagent Preparation

7.5.1 Before beginning the assay, prepare reagents and allow all kit components to reach room temperature. Store unused microwells in the sealed foil pouch with desiccant and all unused reagent in closed containers at 2-8°C.

7.5.2 Wash Solution Preparation: Add 1.0 mL of Wash Solution Concentrate to 100 mL of distilled water. Label the container. This volume is sufficient to wash 40 wells. Wash solution is stable for 30 days at room temperature.

7.5.3 Substrate Preparation - At the time the assay is initiated, add 1 tablet of Reagent B- Substrate Tablet to 5.0 mL of Reagent C-Substrate Diluent. Allow at least one hour for tablets to dissolve. Label container. This volume is sufficient for 48 wells. Prepare only the amount of Substrate needed for immediate use. DISCARD UNUSED SOLUTION.

7.6 Reader Preparation

7.6.1 Fit microwell plate reader with 405 or 410 nanometer (nm) filter.

7.7 Enzyme immunoassay Procedure

7.7.1 Fit the required number of microwells into the holder, allowing for 2 positive controls and 1 blank. Reseal unused microwells. Carefully record Positive Controls, Blank and Sample positions in holder.

7.7.2 Inactivated test broths should be equilibrated to 25 - 37°C prior to running the assay. Do not mix inactivated test broths (from 7.4.2). A new pipette tip must be used for each sample.

7.7.3 Pipette a 100 microliter (µL) inactivated broth into each microwell. Avoid food particles in the liquid transferred. Vortex the Positive Control and pipette 100 µL into each Positive Control well. LEAVE BLANK WELL EMPTY.

7.7.4 Cover microplate and incubate 30 min. at 35°-37°C. Do not stack anything on top of the microwell holder during incubation. Do not agitate plate during any incubation step.

7.7.5 Washing Procedure; following incubation, wash each well three times using one of the options as follows:

1. Completely remove contents of well with a microwell washer. Immediately fill wells with 250 µL of Wash Solution.
2. It is acceptable to remove contents of wells by inverting and vigorously tapping the plate. Wash wells by filling each well with wash solution using a precleaned wash bottle.

Repeat twice for a total of three aspiration/wash cycles per step. Avoid overfilling wells to prevent antigen carry-over to adjacent non-reactive wells. Avoid under filling wells to prevent ineffective washing. Effective washing is critical to obtaining accurate data. Remove excess wash by inverting wells and tapping prior to proceeding to the next step.

7.7.6 IImmediately following aspiration of the third wash, invert the bottle of Conjugate to gently mix. Add 100 µL to each well, including Control and Blank wells. Cover and incubate 30 min. at 35^o -37°C.

7.7.7 Following incubation, aspirate and wash each well three times. Refer to (7.7.5) for Washing Procedure instructions.

7.7.8 Immediately following aspiration of the third wash, gently mix and add 100 µL Substrate to each well, including Control and Blank wells. Cover and incubate 30 min. at 35°C - 37°C. After incubation DO NOT wash wells. Proceed directly to (7.8).

7.8 Reading Results

7.8.1 Immediately following incubation, read and record results.

7.8.2 Standardize the reader by reading the BLANK well and adjusting optical density (O.D.) to zero.

7.8.3 Read the sample absorbance of each well, starting with the two Positive Controls, with a microwell plate reader at 405 or 410 nm.

7.8.4 If reading will be delayed, add 50 µL of Stop Solution to each well. Read within one hour.

7.9 Interpretation of Test Results

7.9.1 Control Value: The Positive Control absorbance values should be greater than or equal to 0.8 O.D. units. Absorbance values below 0.8 O.D. units may indicate problems with the Washing Procedure. Contact BioControl Technical Services for more information.

7.9.2 Cutoff Value: Calculate the mean value of the two Positive Control absorbance readings (O.D. units) and multiply by 0.25 to establish the Cutoff Value:

where PC1 & PC2 = Positive Control absorbance values (O.D. units). Include Positive Controls for each test run.

7.9.3 Positive Results: Samples with absorbance readings greater than or equal to the Cutoff Value are presumptively positive. Positive samples should be confirmed.

7.9.4 Negative Results: Samples with absorbance less than the Cutoff Value are negative.

7.10 Confirmation of Positive EIA Samples

Samples with readings greater than or equal to the cutoff value are presumptively positive. Positive samples must be confirmed using the culture method described in MFLP-80. Use the sample that was retained in 7.4.1.

8. References

8.1 Feldsine, P.T., S. T. Green, A.H. Lienau, and D.E. Kerr. 2005. Comparative Validation Study to Demonstrate the Equivalence of a Minor Modification to AOAC Methods 996.09, VIP® for EHEC and 996.10, Assurance EIA® EHEC with Reference Culture Method for the Detection of Escherichia coli O157:H7 in Beef. J. AOAC Int. 88 (4): 1193-1196.

8.2 Feldsine, P.T., D.E. Kerr, S.C. Leung, A.H. Lienau, R.F. Moser, and L.A. Mui. 2002. Visual Immunoprecipitate Assay Eight Hour Method for Detection of Enterohemorrhagic Escherichia coli O157:H7 in Raw and Cooked Beef (Modification of AOAC Official Method 996.09): Collaborative Study. J. AOAC Int. 85 (5): 1029-1036.

8.3 Feldsine, P.T., D.E. Kerr, S.C. Leung, A.H. Lienau, S.M. Miller, and L.A. Mui. 2002. Assurance® Enzyme Immunoassay Eight Hour Method for Detection of Enterohemorrhagic Escherichia coli O157:H7 in Raw and Cooked Beef (Modification of AOAC Official Method 996.10): Collaborative Study. J. AOAC Int. 85 (5): 1037-1044.