Validation of the Reveal® 3-D for Peanut Lateral Flow Test: AOAC Performance Tested MethodSM 111901

Abstract Background Reveal® 3-D for Peanut is an immunochromatographic, lateral flow test for qualitative detection of peanut residue in food manufacturing and food preparation settings. The test can detect low ppm levels of peanut in clean-in-place (CIP) rinses and in swabs from environmental surfaces and can serve as a tool in managing allergen risk. Objective The objective of the study was to validate the lateral flow method for detection of peanut in CIP rinses, specifically water, peroxyacetic acid/hydrogen peroxide, and quaternary ammonium compound rinses, and in swabs taken from stainless steel and plastic surfaces. Methods CIP rinses spiked with low levels of peanut were tested, as were surfaces inoculated with peanut. Specificity and assay interference were assessed in testing of food commodities with and without added peanut. Assay robustness and test kit stability and consistency testing were also performed. Results Results demonstrated that the lateral flow test can detect peanut in CIP rinses in the range of 2–4 ppm and in environmental surface swabs in the range of 3–4 µg/100 cm2. Results of specificity testing with 29 common food items showed lack of cross-reactivity, and potential assay interference only from walnut. Data from stability trials supports expiration dating for the kit of up to 23 months post-manufacture. Conclusions and Highlights The lateral flow test is a sensitive, specific, and rapid method for detection of low levels of peanut residue in CIP rinses and environmental samples and can be an important component in a comprehensive allergen risk management program.


Introduction
Peanut allergies are one of the most common causes of severe allergic incidents and the most common cause of food-induced anaphylaxis. These reactions can be caused by direct contact to the skin or digestive system, including through ingestion of products cross-contaminated with peanut or through inhalation of dusts or aerosols containing peanuts. When a severe reaction occurs, administration of epinephrine, for example using an EpiPen V R , and emergency care are required (1). Reveal V R 3-D for Peanut is an immunochromatographic, lateral flow test device for detection of peanut residue in clean-in-place rinses and swabs from environmental surfaces. The test is intended for use in an industrial food manufacturing or preparation context. It can be used to validate the adequacy of cleaning and/or to identify problem areas such as buildup of peanut residue in processing equipment, filler heads, etc. In rinses, the test can detect peanut residue at 5 ppm. From environmental swabs, 5 mg/100 cm 2 can be detected. The test is not intended for use in a home or restaurant by peanut-allergic individuals.

Definitions
(a) Probability of detection (POD).-A statistical analysis that measures the proportion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte level or concentration (2,3).

Principle of the Method
Reveal 3-D for Peanut is a lateral flow, immunochromatographic test for detection of peanut proteins. A sample is introduced to the test device where peanut protein (antigen) binds with colloidal gold-conjugated anti-peanut antibodies. The resulting complex flows along a membrane where it encounters a test line coated with anti-peanut antibodies. As the complexed gold particles accumulate, a visible red line is formed. An overload line is included to indicate conditions of antigen excess in the test sample. The overload line is an immobilized mixture of peanut proteins. In the absence (or presence of trace amounts) of peanut in a sample, the anti-peanut gold conjugate binds to the immobilized proteins and forms a visible line. When large amounts of peanut are present in a sample, the antibody gold conjugate will bind the peanut proteins and is inhibited from binding to the overload line.

Test Sample Preparation
Rinse samples (a) Carefully tear/cut or uncap a sachet or tube of Type 9 extraction buffer and add the entire contents to a sample tube. (b) Add 0.25 mL of test sample to the tube containing extraction buffer. (c) Secure the tube cap and shake for 1 min.
Swab samples (a) Carefully tear/cut or uncap a sachet or tube of Type 9 extraction buffer and add the entire contents to a sample tube. For dry surfaces: Remove a sterile swab from the packaging and wet with two drops of swab wetting solution. Swab a 10 x 10 cm area using a crosshatch technique and revolving the swab on the surface. Repeat this swabbing procedure using movements at a right angle to those used in the first swabbing. For other sampling sites (e.g., filler heads) swab as appropriate for the particular location. (b) For wet surfaces: Remove a sterile swab from the packaging. Swab a 10 Â 10 cm area using a crosshatch technique and revolving the swab on the surface. Repeat this swabbing procedure using movements at a right angle to those used in the first swabbing. For other sampling sites (e.g., filler heads) swab as appropriate for the particular location. (c) Return the swab to the sample tube containing extraction buffer and carefully break off the moistened end at the prescored mark so that it remains in the tube. Note: It is essential to place the test device flat on a level surface as soon as liquid has entered the test window to stimulate flow through the device. The test devices are pre-striped with a pale green loading dye in positions T (test), O (overload), and C (control). The loading dye assists with quality and manufacturing checks and does not impact test performance. The loading dye is removed from the test window as the sample flows through the device.

Interpretation of Results
Read the result after 5 min of development. Observations after 6 min may be inaccurate due to overdevelopment of the test.

Further Testing
If desired, samples producing positive Reveal V R 3-D for Peanut results may be tested with a validated laboratory assay (e.g., Veratox V R for Peanut Allergen, Neogen Corp., or equivalent) to obtain quantitative results.

Validation Study
Elements of the validation study included a selectivity study, an interference study, a matrix study, a method robustness trial, lot-to-lot consistency and stability testing, and an independent laboratory study to verify select elements of the internal studies. The study was conducted in accordance with the AOAC ISPAM Guidelines for Validation of Qualitative Binary Chemistry Methods (4).

Preparation of Peanut Spiking Solution
NIST SRM 2387 (peanut butter) was used to prepare peanut spiking solution. Twenty-five milligrams of peanut butter were weighed directly into a 50 mL conical bottom polypropylene tube. Twenty-five milliliters of 10 mM phosphate-buffered saline was added and the tube vortexed at high speed for 1 min to create a uniform suspension. Further dilutions of the 1 mg/mL suspension were made in 0.25% bovine gelatin. For inoculation of environmental surfaces, an alternative procedure was used for preparation of the spiking solution. Fifty milligrams of NIST peanut butter was weighed directly into an extraction bottle. One scoop of extraction additive (Veratox kit) was added followed by 125 mL of 60 C 10 mM PBS. The solution was shaken at 150 rpm in a 60 C water bath for 15 min. The material was allowed to settle for 5 min, then filtered by pouring a minimum of 5 mL through a Whatman #4 filter. Further dilutions were made in 0.25% bovine gelatin.

Selectivity and Interference by Non-Target Food Materials
(a) Methodology.-The selectivity study was designed to demonstrate that the lateral flow method does not produce positive results with other common food ingredients (cross-reactivity), and also to demonstrate that the method is able to detect peanut in the presence of other food ingredients (lack of interference). Twenty-nine non-target food materials were tested, alone and also with 10 ppm added peanut (Table 1). Food materials were pre-screened for natural contamination with peanut using the Veratox for Peanut Allergen ELISA. Food materials were extracted following instructions for the Veratox V R for Peanut Allergen. Five grams of matrix was weighed into an extraction bottle, one level scoop of extraction additive was added, followed by 125 mL of 60 C extraction solution. The bottle was shaken in a 60 C water bath at 150 rpm for 15 min, then allowed to settle for 5 min. A minimum of 5 mL of the extract was then filtered through Whatman #4 filter paper. For each food material, two samples were prepared, one as is, and the other spiked with peanut at 10 ppm. Samples were blind-coded and randomized, then extracted and tested in duplicate following the lateral flow method protocol. Twelve test portions were also tested with the Veratox assay with results ranging from 0.5 to 14.3 ppm peanut. Although a low level of cross-reactivity of cashew in the Reveal assay cannot be ruled out, these results are most consistent with a low level of peanut contamination of the cashew samples. This is supported by the variability in results between test portions observed with both the Reveal and Veratox assays. Only one food product, walnut, showed interference with the ability of the assay to detect peanut. In walnut samples spiked with 10 ppm peanut, both assays produced negative results. Tests were conducted in which the walnut extract was diluted 1:10, spiked with 10 ppm peanut, and then tested. Duplicate assays were positive, showing that the interference can be avoided with dilution of the test sample. Assay interference would only be expected if the product tested contained >10% walnut by weight. In this case, 1:10 dilution of the extract prior to assay is indicated.
Interference by Clean-in-Place Sanitizer (a) Methodology.-An experiment was conducted to assess interference from a clean-in-place sanitizer applied to an environmental surface. Working strength quaternary ammonium sanitizer (Quat-Stat TM 5, Betco, Bowling Green, OH; contains dioctyl dimethyl ammonium chloride and alky-dimethyl benzyl ammonium chloride, used at 0.5 oz per gallon of water) was applied to 10 Â 10 cm stainless steel surface areas and allowed to dry for approximately 1 h. The surface areas were swabbed, and the swabs extracted according to the standard procedure. Extracts were spiked with peanut at levels ranging from 0 to 20 ppm. Triplicate extracts were prepared at each level and assayed. (b) Results.-Results are shown in Table 2. All tests conducted on spiked extracts were positive. Line intensity increased with increasing peanut concentration. There was no evidence of assay interference from the CIP rinse.

Matrix Study
(a) Methodology.-CIP rinses: Quat-Stat 5 was prepared at 25% working strength in water. TexCide TM (Texwipe, Kernersville, NC; contains 5.9% peroxyacetic acid and 27.3% hydrogen peroxide, 4 oz per gallon of water working strength concentration), was prepared at 1% working strength in water. Results of preliminary experiments demonstrated that these were the highest concentrations of the two CIP rinses that could be tested directly in the lateral flow assay without inhibiting assay activity (data not shown). Even the 1% concentration represents an excess over what would normally be present after multiple water rinses following use of clean-in-place sanitizers in a food manufacturing or food preparation setting. For each material, 33 replicate test portions were prepared at each of five peanut target concentrations (0, 1-2, 3, 5, and 10 ppm) using the peanut spiking solution. In some cases, a 0.5 ppm peanut level was included. At least one level was expected to produce a fractional positive data set (25-75% of test portions positive). Three test portions at each level were reserved for testing with the Veratox V R for Peanut Allergen assay to verify the spike levels. The remaining test portions were randomized, blind-coded, and assayed with the lateral flow test. Methodology.-Environmental surface swabs: Diluted peanut spiking solution was used to inoculate plastic and stainless steel 10 x 10 cm surface areas. Approximately 0.25 mL of inoculum was applied. For testing of dry surfaces, the inoculated surfaces were allowed to dry for 16-24 h at 20-24 C. For testing of wet surfaces, the inoculated surfaces were allowed to dry for 30-60 min, but were still visibly wet when sampled. For each surface and condition (wet or dry), the following replicate test portions were prepared: 30 at a peanut level of 1 lg/100 cm 2 , 30 at a level of 2 lg/100 cm 2 , five at a level of 5 lg/100 cm 2 , and five controls without peanut. Again, at least one level was expected to produce fractional positive results. The surface areas were swabbed and extracted. The extracts were randomized, blindcoded, and tested with the lateral flow assay.
POD with a 95% confidence interval was calculated per matrix and spiking level using an online calculator (3).
Results.-Environmental surface swabs: Results for environmental surface swabs are shown in Table 4. Swabs taken from wet and dry stainless steel surfaces produced similar results with POD of 0.8-0.9 at approximately 2 ppm peanut and POD of 1 at 3-4 ppm peanut, respectively. With swabs from plastic, POD was >0.9 at 2 ppm peanut and 1 at 3 ppm peanut for both the wet and dry surfaces.  (Table 5). Water was used as the sample material. Ten replicate test portions spiked with 1 ppm peanut served as the positive sample; this spike level was expected to produce a fractional positive data set. Ten replicate test portions of water served as the negative sample. Test portions were randomized, blind-coded, and tested in the lateral flow assay. POD analysis was conducted comparing results at each condition containing variations to the nominal condition.  dry stainless steel surfaces, results were again consistent with those of the internal trials. POD was 0.4-0.6 at 1 ppm peanut and 1 at 2 ppm peanut.

Discussion
Results of the validation study reported here demonstrate that the Reveal V R 3-D for Peanut lateral flow test is a sensitive and specific method for determination of peanut residue in CIP rinses and in swabs from environmental surfaces. For CIP rinses, including water, peroxyacetic acid/hydrogen peroxide (1% working strength), and quaternary ammonium (25% working strength) sanitizers, considering all trials, POD was 1 in the range of 2-4 ppm peanut. For surface swabs from stainless steel and plastic, POD was 1 at 3-4 ppm. Results of the independent laboratory study with CIP rinse and wet and dry stainless steel surfaces were consistent with those of the internal trials.  Specificity testing with 29 common food commodities showed the assay to be very specific, with no clear evidence of cross-reactivity. Walnut was shown to interfere with the assay's ability to detect peanut at 10 ppm, although the interference can be avoided with 1:10 dilution of the sample prior to assay. As long as the CIP rinse or environmental surface swab contains <10% walnut, test kit users can be confident that there will be no interference. Residual quaternary ammonium sanitizer did not interfere with the ability of the assay to detect low levels of peanut in swabs from a stainless steel surface. Results of robustness testing showed that the assay is able to withstand modest variation in multiple operating parameters simultaneously. Results of stability and consistency testing trials support expiration dating for the Reveal V R 3-D for Peanut test kit of at least 23 months.

Conclusions
Results of the validation study demonstrate that the Reveal V R 3-D for Peanut test is an accurate, sensitive, and specific method for qualitative detection of peanut in CIP rinses and environmental surface samples. Further, the test is simple to perform, requiring no specialized equipment and providing results in less than 10 min, including sample extraction. The test provides a powerful tool to food manufacturing and food preparation personnel for management of peanut allergen risk. It is recommended that the Reveal V R 3-D for Peanut test kit be granted AOAC Performance Tested Method status.