ARCHIVED - Neogen Veratox for Almond Kit : Performance Evaluation

Disclaimer : Inclusion of this method in the compendium does not imply endorsement or approval by Health Canada.

Introduction

The purpose of this study was to generate performance data on the Neogen Veratox for Almond kit, a commercial kit for the detection of almond protein in foods, as part of ongoing efforts to evaluate food allergen detection methodologies and further their introduction into the Compendium of Methodologies. A representative almond powder was chosen to be the designated reference material, according to the definition established by the AMC.

A two stage approach was applied to the evaluation. First, reference material representative of almond was used in recovery experiments where selected food matrices were artificially fortified (spiked samples). Later, food samples containing controlled amounts of almond material which have been incorporated into the food sample under pilot manufacturing conditions, will be analyzed (naturally incurred samples). Results of these experiments and the performance of the kit under each of these evaluation conditions will be reported on, as the data becomes available.

Method Evaluated

The Neogen Veratox for Almond Allergen kit

Evaluation Level

Full evaluation under the guidelines developed for the Compendium of Food Allergen Methodologies.

Designated Reference Material

Almond powder obtained from the Food Allergen Research and Resource Program (FARRP) at the University of Nebraska was used as reference material. This almond powder was a blend of several commercial varieties of almond.

Participating Laboratories

Health Canada Food Allergen Research Lab
Banting Building,
Ottawa, ON

Health Canada Western Regional Laboratory
Burnaby, BC

CFIA Quebec Regional Laboratory
Longueuil, QC

CFIA Western Regional Laboratory
Burnaby, BC

Neogen Laboratory
Lansing MI, USA

Food Products Association
Washington D.C., USA

FARRP
Lincoln, Nebraska, USA

Spiking Levels:
Samples were spiked with 0, 6.25 ppm and 15.63 ppm of the almond paste. These levels were found to conform with the evaluation guidelines of the Compendium which call for spiking levels that give a response from the kit of approximately 0, 2 and 5 times the limit of quantitation (LOQ) when applied to the designated reference material.

Spiking Conditions :
A small aliquot of the almond powder was ground up using mortar and pestle into a paste. Spiking was done using a 1 mg/g suspension of the almond paste in a carboxymethylcellulose (CMC) solution based on a method by Trucksess et al, Preparation of Peanut Butter Suspension for Determination of Peanuts Using Enzyme-Linked Immunoassay Kits, Journal of AOAC International, vol.87,2,2004. Samples were left blank, or spiked at one of two levels and given a blind identification code.

Matrices of Interest :
Three different matrices were included in the evaluation: milk chocolate ( Nestle's Aero chocolate bars ), dark chocolate (Hershey's Special Dark chocolate bars) and cookie (Dad's Oatmeal Cookies from Christie). These commodities were chosen based on which matrices would be most likely to contain undeclared almond. The potential for cross contamination with almond at low levels exists when commodities that do not contain almond as an ingredient are manufactured on the same equipment as commodities that do contain almond.

Materials and Resources

Each participating lab was provided with :

• Neogen Veratox for almond kits with all kit components including the kit insert (PDF Version - 55.62 K) with instructions for running the kit
• blind-coded samples (30 of each matrix - 90 samples in total )

Procedure

Spiking Procedure

The spiking levels were chosen in order to obtain a response of approximately 5 ppm and 12.5 ppm (2 and 5 times LOQ) from the Neogen Veratox for Almond kit. The Neogen kit uses a different standard from the reference almond powder for its calibration curve. Therefore it is expected that there will be a discrepancy in the amount of almond spiked into the samples and the amount of almond quantified by the kit. The amount of reference almond powder used for spiking was chosen accordingly. A preliminary investigation showed that 6.25 ppm and 15.6 ppm of reference almond paste were required to obtain a response equivalent to 5 ppm and 12.5 ppm, respectively, with the Neogen standard. Samples were spiked using a suspension of the reference almond paste in a solution of CMC with Bovine Serum Albumin (BSA) and Thimerosal added. The stock almond paste suspension in CMC was prepared at a concentration of 1 mg/g. The stock almond paste suspension was diluted in PBS (7.8 g in a total volume of 250 mL for the 6.25 ppm spike; 19.5 g in a total volume of 250 mL for the 15.6 ppm spike). A 1 ml aliquot of spiking solution was added to each sample. The blank samples were spiked with a solution made by diluting 14 g of the blank CMC suspension in PBS to a volume of 250 mL. A 1 mL aliquot of this mixture was added to the blank samples.

Preparation of Samples

The 630 samples required (10 replicates at each of 3 levels in 3 commodities for 7 laboratories) were prepared at the Health Canada's Food Allergen Research Laboratory. Samples of 5 g were weighed out into 250 ml screw cap bottles (210 samples for each commodity). The samples were separated into groups of 70, then each group spiked at one of the three spiking levels. Each sample was given a code number, then the samples were grouped together for each of the seven participating laboratories and distributed.

Sample Extraction and Analysis

Each laboratory extracted the samples following the extraction procedure outlined in the Neogen Almond Kit instruction. The extraction was performed directly in the sample bottles provided. The sample extracts were then analyzed using the Neogen Veratox for almond kits following instructions provided in the kit insert. Results were calculated using the Neogen's Log/Logit software.

Results

A number of issues were encountered during this validation. The first involved the cookie samples. When the spiking solution was added to the cookie samples it formed a gummy mass which did not break up sufficiently upon shaking. This resulted in recovery results for the cookie which were slightly lower than expected. The difficulty with recoveries in the cookie samples seemed to be related to the amount of time between spiking and samples extraction, with the lowest recoveries in the samples with the longest time between spiking and extraction. The second issue involved the shipment of samples to Lab#6. These were delayed during transportation and customs clearance for two weeks before finally arriving at their destination. When they were finally analyzed the results from the cookie samples were very poor and were therefore excluded from the study. Three of the 6.25 ppm cookie samples from lab#2 were also excluded due to a malfunction with their shaking apparatus.

The results for the remaining 597 samples showed good inter and intra laboratory consistency. No false positives were reported. Six out of the one hundred and ninety seven 6.25 ppm spikes gave negative results based on the 2.5 ppm cut-off for positive samples. It should be noted that, although below the 2.5 ppm cut-off, all of these samples gave results close to 2.5 ppm and had optical densities considerably higher than the blank sample. Five of these false negatives were in dark chocolate and one in cookie. All of the 15.63 ppm spikes gave positive results.

For each group of 10 samples at one of the three spiking levels in a particular matrix analyzed at the same lab ( for example the 6.25 ppm milk chocolate samples from lab#2 ) a mean and standard deviation were calculated. Any results more than two standard deviations from the mean were considered an outlier and were excluded. Out of 597 samples only six samples were excluded for this reason.

The Z score for an item indicates how far, and in what direction, that item deviates from its distribution's mean, expressed in units of its distribution's standard deviation. A Z-score of 2.0 indicates a result that was two standard deviations above the mean, while a score of -2.0 would indicate a result two standard deviations below the mean. Z-scores were calculated for each lab at each commodity and spiking level. Only three results were above 2.0 and most were below 1.0, which shows a good deal of agreement between all the participating labs.

A summary of the results from each of the seven labs is presented here.

It should be noted that the amount spiked is listed in ppm of almond powder from FARRP, but the response from the kit is in ppm of Neogen standard. Approximate responses of 5 ppm and 12.5 ppm of the Neogen standard were expected for spikes of 6.25 and 15.63 ppm of the almond powder from FARRP.

Conclusion

The Neogen Veratox for almond kit has delivered satisfactory results for the matrices and at the levels tested in this evaluation. Dark chocolate had the lowest recoveries. This was not unexpected as dark chocolate contains high levels of tannins and other phenolic compounds which can interfere with the extraction of proteins. Milk chocolate, which contains less phenolic compounds than dark chocolate, had the best recoveries. Cookie results were in between.

The data from this evaluation, based on spiking samples with the almond powder from FARRP, would be complemented by other data based on "naturally incurred" samples which were manufactured using a known amount of almond. This kind of study is being planned. Data from future studies will be added to this report as it becomes available, in order to further document the performance of this commercial test kit.