Transcript Food Allergies and Intolerances and Their Importance to

Food Allergen Detection Methods:
Advantages and Limitations of
Existing Methods
Joe Baumert, Ph.D.
Food Allergy Research & Resource Program
University of Nebraska
[email protected]
www.farrp.org
Uses of Allergen Tests
• Check raw materials/ingredients for residues
• Assess effectiveness of allergen control
program
• Check finished product for label compliance
• Investigate/confirm consumer complaints
• Regulatory agency efforts
Allergen Analysis
• The methods should measure the most
appropriate analyte – allergen or protein
• The methods should be sufficiently sensitive –
low ppm (2.5 – 10 ppm)
• Users should understand the strengths and
limitations of the analytical methods and choose
the most appropriate method to meet their needs
Key Issues:
Allergen Testing - Specificity
• What Does This Test Actually Measure?
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Allergenic activity
Allergenic epitope
Allergen
Protein from the specific source
General protein
Surrogate marker (DNA, ATP, etc.)
Key Issues:
Allergen Testing - Sensitivity
• Quantitative vs. Qualitative
• Correlation to actual allergen levels
(surrogates)
• Detection limit (low ppm or lower?)
• Limit of Quantitation
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Standard curve issues
Key Issues:
Allergen Testing - Other Factors
• Cost
• Ease of Use (industrial settings)
• Matrix effects
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Processing, solubility, extractability,
interference, cross-reactivity,
comparative stability (surrogates)
Detection Methods
• Enzyme Linked Immunosorbent Assay (ELISA)
• General Protein Tests
• ATP/Bioluminescence Tests
• Polymerase Chain Reaction (PCR)
• Mass Spectrometry Methods (LC-MS/MS)
ELISA and ELISA-Based
Technologies
• Include ELISA kits, lateral flow
devices (dipsticks), swabs
• Current state-of-the-art
• Specific
• Sensitive
• 5 min-6 hr analytical process
• Suitable for food-processing settings
Source: microscopesblog.com
ELISA and ELISA-Based
Technologies
• Specific – detects protein(s) from source; not
always specific for allergenic protein
• Sensitive (low ppm and could be less)
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FARRP/Neogen methods – Limit of Quantitation (LOQ) of
1-2.5 ppm
No clinical reason to “chase molecules”
• Quantitative (96 well) and Qualitative formats
(lateral flow and swab) formats
ELISA and ELISA-Based
Technologies
• Not cheap
• ELISA kit usually $250-650 USD
⁻ Can test ~10-38 samples per plate
• Lateral flow devices (strip tests) ~ $15 per stick
• Contract labs charge $50-200 USD per sample
Commercial ELISAs
• Peanut
• Soybean
• Milk
• Crustacea
• Egg
• Mustard
• Gluten
• Lupine
• Almond
• Sesame seed
• Hazelnut
• Buckwheat
• Walnut
FARRP Confidential Analytical
Testing: ELISAs
Fully Developed
• Peanut
• Milk
• Egg
• Processed Soy
• Soy Flour
• Almond
• Hazelnut
• Shrimp Tropomyosin
• Lupine
*In-house ELISAs
• Sesame
• Gluten/Gliadin (wheat,
barley, rye)
• Buckwheat*
• Walnut*
• Mustard
• Clam*
• Pecan*
• Cashew*
In Development
• Pistachio**
• Fish
**in use for analysis
All ELISAs Are Not Created Equal
• Specificity
• Sensitivity
• Format
• Quantitative vs. Qualitative
ELISA Points of Difference
• Antibody Specificity – total protein vs. allergen
• Polyclonal vs. Monoclonal Antibodies
• Calibrators
• Effects of Processing on Detection
• Extraction Methods
• Sensitivity Limits
ELISA Specificity
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Total Peanut vs. Ara h 1
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Total Milk vs. Casein vs. β-Lactoglobulin
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Soy Flour vs. Processed Soy
ELISA Sensitivity
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The limit of quantitation may appear to be the
same, but what units does the test kit report in?
– Neogen Veratox Total Milk – 2.5 ppm NFDM
– R-Biopharm FAST Milk – 2.5 ppm milk protein
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NFDM is approximately 35% milk protein
– R-Biopharm FAST Milk LOQ = 7.1 ppm NFDM
– ~3-fold difference in sensitivity
Using ELISAs for Analysis
• What do you want to measure?
⁻ Select appropriate detection system according to major
components in the product
 Example: Milk
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Neogen: Total Milk
r-Biopharm: β-lactoglobulin
ELISA Systems: Casein
• What standards are used in the method?
⁻ NFDM, Casein, β-lactoglobulin
Things You Can Test With ELISAs
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CIP rinse water
Equipment surfaces
Environmental surfaces
In-process product (“throwaway”)
“Push-through” – product, ingredient, etc.
– Ice, flour, other things used to “scour” equipment
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Individual ingredients
Finished products
Using ELISAs for Sanitation
Validation/Assessment
• Visually clean is the standard in the food industry
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If residue is present on the equipment surface, a positive ELISA is likely
No need to test – clean again
• Visual inspection can be quite effective
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Verification and validation of allergen removal using specific ELISA
methods is needed
Swabbing equipment surfaces (contact surfaces and hard to reach areas)
or testing CIP final rinse water is commonly done for allergen assessment
Using ELISAs for Sanitation
Validation/Assessment
• Recommended to use swabs provided by ELISA kit
manufacturers
– Microbial sponges/swabs can have media that can interfere with
the ELISA test
– Allergenic proteins may not be readily extracted from some swab
types, sponges, Q-tips
• Use aseptic technique when collecting swab
samples
– Package swab sample individually
– Do not carry opened swabs in shirt pocket throughout processing
facility
Swab/Dipstick Testing Issues
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For swabs, always swab a specific area size for
smooth surfaces
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Also, select areas that are hard to clean (crevices,
cracks, welds, joints, etc.)
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Ideal for surface testing
But can be used for qualitative testing of product,
ingredients, etc.
Swab/Dipstick Testing Issues
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Quantifying the results – a positive swab does not
mean that the product will be contaminated in every
case
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But a positive swab means surface not clean
Raw Material Testing
• Check for farm-level contamination
(e.g. soy in wheat, pecans in peanuts)
• Check for contamination during transport and storage
• Check of cross-contact at supplier level
• Assess effectiveness of supplier allergen control
program
• Determine adherence to purchasing specifications
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Finished Product Testing:
Testing of some finished product is recommended
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Only test finished product when your cleaning and
sanitation procedures are validated and you are
99.99% sure that you will not find a positive
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This is especially important if marketing products with “Free”
Statements
Ex. Gluten Free, Peanut Free
Do not want to test yourself into a recall situation
If making a ‘free’ claim, ensure that the most sensitive
analytical method is used
Things You Cannot Analyze with Allergen ELISAs
• Hydrolyzed or fermented ingredients
• Oils or Oil-Derived Ingredients
‒ Lecithin, tocopherol, phytosterols
• Retorted Products:
- Proteins may be insoluble
- High pressure / high heat
- Structural changes
• Salad Dressing:
- Proteins may be precipitated (insoluble?) due to pH

Run a positive control to determine if the ELISA method is
suitable
Other Tests We
Get Questions About
(Surrogate Tests)
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General Protein Tests
3M™ Clean-Trace ™ Surface Protein (Allergen)
– Swab method for detection of protein from environmental samples
– Based on biuret/BCA reaction
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Detects protein regardless of source but not specific for allergenic
protein
– Cannot determine if a positive result is due to an allergenic protein or nonallergen
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Detection Limit ~ 10 – 50 µg protein
Can be used for routine sanitation verification but should first
validate the cleaning methods with specific ELISAs
ATP/Bioluminescence Tests
• None are specific for allergens
• ATP levels vary between foods
• Does not prove presence of protein
– Protein makes the problem with allergens
• Have not been shown yet to correlate with
Source: sigmaaldrich.com
specific ELISA tests in research so far
• Don’t let a salesman tell you an ATP tests mirrors the
allergen level – it doesn’t
PCR Methodology
• Specific – to the source (DNA) but not to the allergenic
proteins
• Sensitive (very)
• Semi-quantitative
• Depends on specific primers
• Available for many allergenic food sources
Source:
scienceblogs.c
om
PCR Methodology
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PCR (DNA) tests available – U.S. industry not as
interested in using PCR for allergenic residue detection
– Not practical for in-plant use
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expensive equipment required (>$30,000 USD)
isolated lab required to avoid contamination
– Does not prove presence or absence of
protein/allergen
Limitations of PCR
• These cannot be differentiated by PCR
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DNA differentially distributed as compared to
proteins
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Beef/milk
Egg/chicken
Ex. DNA in low abundance in milk, egg white, and in some protein
concentrates and isolates
Protein levels are in high quantities in these products
Could give rise to false negative results
Food matrix and processing can negatively
impact the extraction and detection
Mass Spec Methods
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Specific to a particular protein or peptide
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Proteins are enzymatically digested (trypsin)
– Careful section of signal peptides of interest needed for
accurate determination of potential allergens present
– Section based on abundance and stability of peptides for a
particular allergen
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Sensitive but not more sensitive than ELISA
Currently used as a confirmatory method/research tool
Mass Spec Methods - Advantages
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Can detect several allergens simultaneously
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Detection of allergens is not affected by structural
changes or modifications
– Not dependent on proper folding of protein
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No false positive results due to cross-reactivity
– But must find reliable signal peptide
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A wider range of extraction methods can be used
Mass Spec Methods - Limitations
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Food matrix and processing can negatively
impact the extraction
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Limited information on the effects of the food
matrix on detection
– Quantitative analysis is limited currently
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Requires expensive equipment and an expert
analyst to interpret results
– Cannot be used for routine, in-plant testing
How To Decide What Test to Use in a
Specific Situation?
• The specificity of the test method is one concern –
protein, allergenic source protein, DNA, ATP, MS
• The sensitivity of the test method is a second concern;
will the method support the corporate target level?; or
can you correlate the method results to the corporate
target level?
How To Decide What Test to Use in a
Specific Situation?

Recommended to validate removal of allergenic
residue using specific ELISAs
– ATP and general protein tests do not detect proteins from
allergenic sources specifically so the effectiveness of these
tests ALONE as the sole approach must be carefully
examined

Surrogate testing (protein, ATP) can be helpful in some
cases
– ATP or general protein swabs can provide a good quick
check on sanitation effectiveness during routine cleaning
How To Decide What Test to Use in a
Specific Situation?
• Allergen-specific ELISAs (either swabs or dipsticks) will
allow very specific and sensitive detection of allergenic
residues
• The ELISA kits only detect specific proteins or allergens
from the source and are quantified in ppm which allows
quantitative risk assessment
• If nothing is detected, the product or surface is quite
safe from an allergen perspective