ARCHIVED - Neogen Veratox for Egg 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 Egg kit, a commercial kit for the detection of egg in foods as part of ongoing efforts to evaluate food allergen detection methodologies and their introduction into the Compendium of Methodologies. Due to the nature of egg protein, which changes its structure when heated, the effectiveness of the extraction conditions and the response of antibody-based tests will depend on the state of the egg being detected (raw vs. cooked). For this reason it is difficult for any one standard to be representative of egg in a variety of commodities and states. Nevertheless, it was possible to choose material which could be used as a 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 egg was used in recovery experiments where selected food matrices were artificially fortified. (spiked samples) Later, food samples containing controlled amounts of egg material which had been incorporated in the food sample under pilot manufacturing conditions, were 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 Egg kit.

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

Designated Reference Material : NIST 8415 whole egg powder. This is a reference material made of spray dried raw whole eggs supplied by the National Institute of Standards and Technology in the United States and is different from the standard calibrators provided with the Neogen Veratox for Egg kit.

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

Spiking Levels: Samples were spiked with 0, 25 ppm and 62.5 ppm of the NIST 8415 standard. 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 when applied to the designated reference material.

Spiking Conditions :

Spiking was done using a suspension of the NIST 8415 egg powder 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 spiked at one of three levels and given a blind identification code.

Matrices of Interest :

Three different matrices: pasta, whole wheat bread and ice cream were included in the evaluation. These commodities were chosen based on which matrices would be most likely to contain undeclared egg. If manufactured on the same equipment as similar commodities which do contain eggs the potential for cross contamination with egg at low levels exists. Pasta, bread and ice cream have been involved in previous cases of undeclared eggs.

Materials and Resources

Each participating lab was provided with:

• Neogen Veratox for egg kits with all kit components including the kit insert 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 L.O.Q.) from the Neogen Veratox for Egg kit.

The Neogen kit uses a different standard from the NIST 8415 whole egg powder for its calibration curve. Therefore it is expected that there will be a discrepancy in the amount of egg spiked into the samples and the amount of egg quantified by the kit. The amount of NIST 8415 used for spiking was chosen accordingly. A preliminary investigation showed that 25 ppm and 62.5 ppm of NIST 8415 were required to obtain a response equivalent to 5 ppm and 12.5 ppm with the Neogen kit. Samples were spiked using a suspension of the NIST 8415 egg powder in a solution of CMC with Bovine Serum Albumin (BSA) and Thimerosal added. The stock solution of NIST 8415 in CMC was prepared at a concentration of 1 mg/g. This solution was then diluted in Phosphate Buffer Saline (PBS) (1g in 8ml for the 25 ppm spike, 1g in 3.2ml for the 62.5ppm spike). 1ml aliquots of spiking solution were added to each sample. The blank samples were spiked with a solution made by diluting the blank CMC solution in a ratio of 1 g in 8 ml PBS (as with the 25 ppm spikes but without the NIST egg).

Preparation of Samples :

The 450 samples required (10 replicates at each of 3 levels in 3 commodities for 5 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 (150 samples for each commodity). The samples were separated into groups of 50, 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 five participating laboratories and distributed.

Sample Extraction and Analysis :

Each laboratory extracted the samples following the extraction procedure outlined in the Neogen Egg Kit instruction. The extraction was performed directly in the sample bottles provided. Extraction additive and 125 ml of extraction buffer were added to each sample then the shaking step started. For the pasta samples, the protocol described by the test kit manufacturer was slightly altered. A problem was encountered with the spiking solution, which formed a gummy mass on contact with the blank pasta. When extracting the pasta samples only, participants were asked to add a step after the extraction buffer was added and before the shaking step, consisting of a short period of homogenization using a polytron or similar homogenization device. The sample extracts were then analyzed using the Neogen Veratox for egg kits following instructions provided in the kit insert. Results were calculated using the Neogen's Log/Logit software.

Results

One of the participating laboratories had difficulty with the pasta samples due to the problem with the spiking solution mentioned previously. The data from this laboratory on the pasta samples has been excluded. The results from this evaluation showed good inter and intra laboratory consistency. Of the remaining 420 samples tested there were no false positives reported and no false negatives. 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 25 ppm Pasta samples from lab 4) a mean and standard deviation were calculated. Any results more than 2 standard deviations from the mean were considered outliers and excluded. Out of 420 samples only 9 results were excluded.

Next z-scores were calculated for each lab at each commodity and spiking level. All results were below 2.0 and most results 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 five labs is presented here.It should be noted that the amount spiked is listed in ppm of NIST 8415 powder but the response from the kit is in ppm of Neogen standard. Approximate responses of 5ppm and 12.5 ppm of the Neogen standard were expected for spikes of 25 and 62.5 ppm of the NIST 8415 egg standard.

Conclusion :

The Neogen Veratox for egg kit has delivered satisfactory results for the matrices and at the levels tested in this evaluation.

The choice of the NIST 8415 as the standard for this evaluation is a compromise based on reference materials available at the time of the study. The data from this evaluation, based on the NIST 8415 standard, would be complemented by other data based on naturally incurred samples, or using a different standard. The newly generated results will be added to this report, in order to further document the performance of this commercially available test kit.