Product Number: 8440

Veratox® for Almond

VALIDATION
REPORT
Revision 6, April 2022
 Veratox
for Almond
®

Summary
Veratox® for Almond (NEOGEN® item 8440) is intended for the
full quantitative analysis or simple screening of almond protein
residues in food products such as cookies, crackers, chocolate
bars, ice cream, beverages, cereals, and clean-in-place rinses.
Quantitation with Veratox for Almond ranges from 2.5–25 ppm,
and can be extended by dilution of positive extracts. The
validation report details the findings of the experimental
evaluation to establish product claims for the Veratox for
Almond test.
Limit of detection: The limit of detection was found to be <1.0 ppm
and the limit of quantitation was found to be 2.5 ppm.
Ruggedness: Inter (CV 7.6%) and intra (CV 3.3%) assay variability
was tested across operator, day, lot, and concentration. Results
met acceptance criteria.
Cross-reactivity: Cross reacts with sunflower seed, apple seed,
and common Prunus genus foods (ie. apricot, cherries, peaches,
nectarines, and plums). Cross-reactivity has been identified
with the seed (stone); however the flesh (seedless fruit) does not
cross react.
Market basket screening: A series of various foods were purchased
at local markets and screened for almond allergen content.
Spike and recovery: The Veratox for Almond test kit demonstrated
excellent recovery in a variety of matrices.
Robustness: Variations in the extraction and ELISA procedure
were investigated with no significant effect on the functionality
of the test kit.

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Materials and Methods
All tests were conducted on standard quality control (QC) approved lots of Veratox for Almond test kits. All assays
were performed in accordance with the test kit insert. Spiked samples were spiked and held for 24 hours to allow
spike to fully incorporate into the sample.

Limit of Detection
The mean of ten known negative samples was calculated and the limit of detection (LOD) was calculated by adding
the mean to 3x standard deviation (StDev).

Replicate ppm
1 0.28

2 0.28

3 0.27

4 0.25

5 0.27

6 0.26

7 0.29

8 0.27

9 0.28

10 0.26

Mean 0.27

StDev 0.01

LOD (Mean + 3x StDev) 0.30

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Ruggedness
Inter-assay Variability

0 ppm 2.5 ppm 5 ppm 10 ppm 25 ppm

0.267 0.210 0.587 0.622 0.863 0.929 1.402 1.388 2.154 2.219

0.272 0.284 0.592 0.572 0.887 0.880 1.452 1.375 2.213 2.160

0.295 0.344 0.566 0.587 0.801 0.848 1.205 1.331 1.944 2.081

0.256 0.264 0.551 0.572 0.787 0.813 1.216 1.092 1.919 1.995

0.261 0.287 0.580 0.571 0.838 0.860 1.364 1.298 2.036 1.900

0.287 0.285 0.609 0.576 0.840 0.841 1.102 1.284 2.042 1.780

0.302 0.270 0.535 0.538 0.726 0.791 1.135 1.201 1.831 1.820

0.286 0.265 0.586 0.472 0.795 0.789 1.233 1.239 1.880 1.815

Mean OD 0.277 0.570 0.831 1.270 1.987

StDev 0.028 0.035 0.049 0.110 0.148

% CV 10.0 6.1 5.9 8.7 7.4

Mean CV% 7.6

Intra-assay Variability

0 ppm 2.5 ppm 5 ppm 10 ppm 25 ppm

0.255 0.251 0.508 0.521 0.739 0.751 1.191 1.235 1.941 1.959

0.247 0.249 0.524 0.539 0.755 0.752 1.321 1.237 1.974 1.972

0.238 0.241 0.518 0.516 0.728 0.744 1.335 1.227 1.985 1.990

0.240 0.262 0.521 0.521 0.750 0.768 1.240 1.243 2.032 2.057

0.251 0.245 0.431 0.523 0.746 0.721 1.234 1.211 1.955 1.966

0.245 0.244 0.535 0.537 0.765 0.736 1.344 1.245 2.086 2.088

0.245 0.251 0.532 0.517 0.762 0.736 1.256 1.247 2.009 2.025

0.246 0.251 0.519 0.510 0.752 0.698 1.358 1.231 1.893 1.896
Mean OD 0.248 0.517 0.744 1.260 1.989

StDev 0.006 0.025 0.018 0.050 0.058

% CV 2.4 4.8 2.4 4.0 2.9

Mean CV% 3.3

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Cross-reactivity
Cross reacts with sunflower seed, apple seed, and common Prunus genus foods (ie. apricot, cherries, nectarines, peaches,
and plums). Cross-reactivity has been identified with the seed (stone); however the flesh (seedless fruit) does not cross
react. A panel of 39 additional samples including nuts, grains, proteins, and seeds were also tested and did not
demonstrate any cross-reactivity with the Veratox for Almond test.

No. Commodity No. Commodity No. Commodity No. Commodity

1 Adzuki Bean 11 Coconut 21 Peanut 31 Rye
2 Anasazi Bean 12 Cornmeal 22 Pecan 32 Sesame Seed
3 Beef 13 Great Northern Bean 23 Pepper Seed 33 Skim Milk
4 Black-eyed Pea 14 Hazelnut 24 Pine Nut 34 Split Pea
5 Black Turtle Bean 15 Kidney Vean 25 Pinto Bean 35 Turkey
6 Black Walnut 16 Lentil 26 Pistachio 36 Walnut
7 Brazil Nut 17 Lima Bean 27 Poppy Seed 37 Wheat
8 Cashew 18 Lecithin 28 Pork 38 Wheat Gluten
9 Chickpea 19 Macadamia Nut 29 Rice 39 Wheat Flour
10 Cocoa 20 Navy Bean 30 Rice (Brown)

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Market Basket
Market basket samples with and without almond listed as an ingredient were collected from local markets. All results
above the limit of quantitation (LOQ) were not considered quantitative until further dilutions were made, which is
reflected in the data below.

Commodity Dilution ppm Calculated ppm

Straight 0 0

Vanilla Coffee 1:100 0 0

1:500 0 0

Straight 0 0

Sharp Cheddar Cheese Spread 1:100 0 0

1:500 0 0

Straight >25 >25

Rice Pilaf with Almond 1:1000 >25 >25

1:4000 22.5 90,000

Straight >25 >25

Almond Ice Cream 1:1000 >25 >25

1:3000 13.4 40,200

Straight >25 >25

Almond Biscotti 1:100 >25 >25

1:500 19.7 9861

Straight >25 >25

Belgian Almond Thin 1:1000 >25 >25

1:5000 16.3 81,500

Straight >25 >25

Vanilla Almond Protein Bar 1:1000 >25 >25

1:2000 19.5 38,960

Straight >25 >25

Almond Milk, Organic 1:1000 >25 >25

1:3000 16.1 48,216

Straight >25 >25

Almond Milk, Vanilla 1:1000 >25 >25

1:3000 11.0 33,054

Straight >25 >25

Almond Milk, Chocolate 1:1000 >25 >25

1:3000 12.0 35,967

Note: All results reported as ppm.

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Spike and Recovery
Veratox for Almond with recovery from spiked samples.

0 ppm Almond Added

Almond Recovered
Commodity Replications Avg.
Vanilla Ice Cream 0.1 0.1 0.1 0.1

Cookies 0.3 0.1 0.2 0.2

Pasta Sauce 0.1 0.0 0.1 0.1

Milk Chocolate 0.0 0.0 0.0 0.0

5 ppm Almond Added

Almond Recovered
Commodity Replications Avg. %
Vanilla Ice Cream 5.1 5.0 4.9 5.0 100.0

Cookies 4.3 3.3 3.8 3.8 76.0

Pasta Sauce 5.4 5.4 5.7 5.5 110.0

Milk Chocolate 5.0 4.3 4.4 4.6 91.0

15 ppm Almond Added

Almond Recovered
Commodity Replications Avg. %
Vanilla Ice Cream 9.1 9.7 9.5 9.4 94.0

Cookies 7.9 6.9 7.2 7.3 73.0

Pasta Sauce 9.8 10.9 10.6 10.4 104.0

Milk Chocolate 9.3 8.8 8.6 8.9 89.0

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Robustness
Incubation Time
The recommended incubation time in minutes is 10:10:10. Studies were performed with less, more, and
variable incubations to demonstrate the effect of timing.

0 ppm Almond Allergen Added 5 ppm Almond Allergen Added 15 ppm Almond Allergen Added
Almond Recovered
Day 1 Times Replications Avg. Replications Avg. % Replications Avg. %
8:8:8 Minutes 0.0 0.0 0.1 0.0 6.2 5.4 5.7 5.8 115.3 18.1 15.3 18.1 17.2 114.4

10:10:10 Minutes 0.3 0.3 0.1 0.2 5.6 5.7 4.7 5.3 106.7 16.3 16.7 14.7 15.9 106.0

12:12:12 Minutes 0.4 0.4 0.3 0.4 5.2 5.2 5.4 5.3 105.3 15.4 16.5 16.1 16.0 106.7

8:10:12 Minutes 0.0 0.0 0.1 0.0 5.5 5.4 5.7 5.5 110.7 15.6 15.6 16.7 16.0 106.4
Day 2 Times Replications Avg. Replications Avg. % Replications Avg. %
8:8:8 Minutes 0.0 0.0 0.0 0.0 4.5 4.1 5.1 4.6 91.3 12.8 13.2 14.6 13.5 90.2

10:10:10 Minutes 0.2 0.0 0.2 0.1 4.8 4.5 4.8 4.7 94.0 14.5 13.3 14.6 14.1 94.2

12:12:12 Minutes 0.1 0.4 0.3 0.3 5.0 4.6 5.0 4.9 97.3 15.2 13.8 15.5 14.8 98.9

8:10:12 Minutes 0.1 0.3 0.0 0.1 4.9 4.6 4.8 4.8 95.3 14.4 13.4 14.0 13.9 92.9

Reagent Volume
The recommended reagent volume is 100 µL. This study was performed using 95 µL and 105 µL volumes to
demonstrate the effect of reagent volumes.

0 ppm Almond Allergen Added 5 ppm Almond Allergen Added 15 ppm Almond Allergen Added

Almond Recovered
Day 1 Volumes Replications Avg. Replications Avg. % Replications Avg. %
95 µL 0.2 0.0 0.2 0.1 5.5 5.8 6.2 5.8 116.7 15.8 17.5 18.0 17.1 114.0

100 µL 0 0 0 0 5.9 5.4 5.6 5.6 112.7 16.9 16.7 18.4 17.3 115.6

105 µL 0.4 0 0.2 0.2 5.8 5.4 4.9 5.4 107.3 15.7 15.6 14.3 15.2 101.3
Day 2 Volumes Replications Avg. Replications Avg. % Replications Avg. %
95 µL 0.1 0.2 0.0 0.1 5.4 4.9 5.0 5.1 102.0 13.2 14.7 15.6 14.5 93.7

100 µL 0.0 0.0 0.0 0.0 4.6 4.6 4.3 4.5 90.0 13.1 14.2 14.0 13.8 91.8

105 µL 0.0 0.3 0.1 0.1 4.6 4.8 4.5 4.6 92.7 14.0 12.8 13.2 13.3 88.9

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Shaking Effects
The recommended shake time at the beginning of each incubation is 20 seconds. This study compares no shaking
and a 30 second shake to the standard 20 second shake to demonstrate the effect of shaking.

0 ppm Almond Allergen Added 5 ppm Almond Allergen Added 15 ppm Almond Allergen Added
Almond Recovered
Day 1
Replications Avg. Replications Avg. % Replications Avg. %
Shake Times
0 Seconds 0.5 0.0 0.0 0.2 5.6 5.3 5.3 5.4 108.0 15.7 17.3 16.2 16.4 109.3

20 Seconds 0.2 0 0.2 0.1 4.6 5.4 5.2 5.1 101.3 14.4 15.2 14.2 14.6 97.3

30 Seconds 0.2 0.1 0.4 0.2 4.5 2.3 4.6 3.8 76.0 13.7 13.4 12.7 13.3 88.4
Day 2
Replications Avg. Replications Avg. % Replications Avg. %
Shake Times
0 Seconds 0.0 0.0 0.2 0.1 4.7 4.5 5.0 4.7 94.7 13.6 12.9 14.1 13.5 90.2
20 Seconds 0.0 0.0 0.4 0.1 4.5 4.5 4.9 4.6 92.7 13.1 12.8 12.0 12.6 84.2

30 Seconds 0.2 0.0 0.0 0.1 4.6 4.4 4.5 4.5 90.0 12.5 12.4 14.4 13.1 87.3

Conclusion
Throughout a wide range of tests, Veratox for Almond demonstrated excellent spike recovery and superior test
robustness. This, coupled with its reproducibility, make Veratox for Almond a good choice for the quick, accurate
detection of almond contamination in a range of food products.

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