ARCHIVED - Elisa Systems Beta-Lactoglobulin: Performance Evaluation
Disclaimer: Inclusion of this method in the compendium does not imply endorsement or approval by Health Canada.
This evaluation involved the analysis of selected food matrices which had been artificially fortified (spiked samples) with the whole milk powder.
The Elisa Systems Beta-Lactoglobulin kit.
Full evaluation under the guidelines developed for the Compendium of Food Allergen Methodologies.
Designated Reference Material:
Whole milk powder obtained from the National Institute of Standards & Technology (NIST) was used as a reference material. The milk powder is joint material of Agriculture and Agri-Food Canada and NIST with a reference material # 8435.
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
Laboratoire d'Hormonologie, CER,
Marloie , Belgium
Samples were spiked with 0, 32 ppm and 80 ppm of the NIST 8435 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 (LOQ) when applied to the designated reference material.
Spiking was done using a suspension of the NIST 8435 whole milk powder in phosphate buffered saline (PBS) solution. 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: Baby Cereal, Cookies and Dark Chocolate. These commodities were included as representatives of some of the matrices which could be most likely to contain undeclared milk proteins. The potential for cross contamination with milk products at low levels exists when commodities that do not contain milk or components of milk as an ingredient are manufactured on the same equipment as commodities that do contain milk or milk components.
Materials and Resources
Each participating lab was provided with:
- Elisa Systems Beta-Lactoglobulin 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 )
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 Elisa Systems Beta-Lactoglobulin kit. The Elisa Systems Beta-Lactoglobulin kit uses a different standard than the NIST Whole Milk Powder for its calibration curve. Therefore it is expected that there will be a discrepancy in the amount of NIST whole milk powder spiked into the samples and the amount of Skim Milk Powder equivalent adopted as a standard by the Elisa Systems test kit. The amount of NIST whole milk powder used for spiking was chosen accordingly. A preliminary investigation showed that 32 ppm and 80 ppm of NIST whole milk powder were required to obtain a response equivalent to 2.5 ppm and 12.5 ppm, respectively, with the Elisa Systems standard. Samples were spiked using a 440 μg/ml working solution of NIST 8435 whole milk powder. The 440 μg/ml working stock solution of NIST 8435 was prepared by serial dilution. A 1g aliquot of the whole milk powder was dissolved in 10 ml of PBS (100 mg/ml), then diluted ( 1:10 ) with additional PBS (10 mg/ml) and, then, 11 ml of (10 mg/ml) was brought to 250 ml. The blank samples were spiked with 1.0 ml of PBS, the 32 ppm samples with 0.364 ml of 440 μg/ml of working solution + 0.5 ml PBS and the 80 ppm samples with 0.909 ml of 440 μg/ml of working solution.
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 in Ottawa. 5 g samples 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 shipped by courier.
Sample Extraction and Analysis:
Each laboratory extracted the samples following the extraction procedure outlined in the Elisa Systems Beta-Lactoglobulin Kit instructions. The extraction was performed directly in the sample bottles provided. The sample extracts were then analyzed using the Elisa Systems Beta-Lactoglobulin kit, following the instructions provided in the kit insert.
Results / Discussion
The results for the 450 samples in the study showed good inter and intra laboratory consistency. No false positives were reported. Six false negatives were reported out of the 150 lower level spiked samples. All six of these were reported in the dark chocolate samples. It should be noted that, although below the 2.5 ppm LOQ listed in the kit insert instructions, all of these samples gave results close to 2.5 ppm and had optical densities (OD) considerably higher than the blank sample. All of the higher level spiked samples 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 32 ppm dark 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 450 samples only 8 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 the majority were below 1.0, which shows a good agreement between all the participating labs. The highest z-scores were obtained for the blank samples, which is understandable since small variations in O.D. are proportionally larger for the blanks than for the other samples.
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 whole milk powder, but the response from the kit is in ppm of the Elisa Systems standard, expressed in ppm of skim milk powder. Approximate responses of 5 ppm and 12.5 ppm of the Elisa Systems standard were expected for spikes of 32 ppm and 80 ppm of the NIST whole milk powder. The choice of the NIST whole milk powder was made in order that a well characterized and readily available material was used as the reference material for this evaluation. Since the calibrator supplied with the kit (commercial, skim milk powder) is different from the material used for spiking (NIST, whole milk powder) the difference in response of the kit to the two materials was expected.
The ElisaSystems Beta-Lactoglobulin 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. Cereal had the best recoveries.
The data from this evaluation, based on spiking samples with the whole milk powder from NIST, may be complemented by other data based on "naturally incurred" samples which were manufactured using a known amount of milk or Beta-Lactoglobulin. Data from any future studies involving these kinds of samples will be added to this report as it becomes available, in order to further document the performance of this commercial test kit.
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