Analytical Methods for Allergens

Sue Hefle, Ph.D.

Associate Professor and Co-Director
Food Allergy Research and
Resource Program (FARRP)
University of Nebraska
shefle1@unl.edu
www.farrp.unl.edu
402-472-4430
Food Allergy Research and Resource Program 2005
 Basics of Analytical Methods for
Allergens
Most used is ELISA-based (includes lateral flow)
Most successful kits use polyclonal antibodies, but
occasional kit uses monoclonal antibodies directed
against a single protein
Usually, antibodies are directed against a crude extract
of allergenic food
Not necessary to measure allergen; industry really just cares if
any peanut is there, not if Ara h 1 is there!
Challenge: different standards used in different kits; also
different antibodies

Food Allergy Research and Resource Program 2005

 Basics of Analytical Methods for
Allergens
Detection limits range from 0.1 2.5 ppm for
quantitative methods
Using a method that has a very low detection limit:
Every kit has the ability to have a low detection limit
Clinical relevance vs. chasing molecules around a food plant
(paralysis by analysis)
Adversely affects the quality of life for food-allergic
individuals because of the industry reaction in the form
of increased use of may contain-type labeling
Current detection limits have worked very well for 7
years
Food Allergy Research and Resource Program 2005
 Status of Allergen
Testing in the U.S.
Many companies are testing for allergen residues
ELISA or lateral flow is the preferred method
Some do in-house testing, others use contractor labs
Most companies are not testing finished product
Are testing to validate sanitation methods; environmental swabbing
Some testing of finished product done when product is under full control
PCR(DNA) tests available FARRP does not recommend for
allergenic residue detection
Not practical for in-plant use; expensive equipment and isolated lab
required
Does not prove presence or absence of protein/allergen
ATP tests do not correlate completely with allergen ELISA results

Food Allergy Research and Resource Program 2005

 Peanut Detection Kits
Three peanut ELISA kits have been performance tested by FDA through
AOAC-RI
Neogen
R-Biopharm
Tepnel
Five peanut ELISA kits have been studied in one JRC interlab trial
Neogen
R-Biopharm
ELISA Technologies
Tepnel
Pro-Lab Diagnostics
Two peanut lateral flow devices are currently in a JRC interlab trial
Neogen
Tepnel
Only one matrix, though - cookies

Food Allergy Research and Resource Program 2005

 Validation of Kits
FDA-AOAC have said they plan more validation studies
with other test kits; this has been the case for more than
2 years with no apparent progress
U.S. food industry and other regulatory agencies (i.e.
Canada) have moved way ahead of FDA/AOAC
U.S. industry has been testing for 7 years, since first peanut test
came out, and has increased the amount of testing each year
Health Canada/CFIA Compendium of Food Allergen
Methodologies; commercial and in-house methods
http://www.hc-sc.gc.ca/food-aliment/cs-ipc/fr-
ra/e_allergen_compendium.html

Food Allergy Research and Resource Program 2005

 Validation of Kits
More JRC trials likely
Other groups planning interlab trials, some
with model foods

Food Allergy Research and Resource Program 2005

 Validation of Kits
Kit companies do much more extensive
validation than will ever be done by any
regulatory agency or academic center
Have liability issues, reputation issues

Food Allergy Research and Resource Program 2005

 Reference Materials and Model
Foods
Reference materials
Not many available; REALLY needed
NIST is one source, but NIST standards were not made for allergen testing
and often do not represent the type of allergenic materials used in the food
industry
Standard used in AOAC-RI-FDA study = peanut butter; varieties not known with
certainty (manufacturer would not divulge to FDA); different peanut varieties
have different responses in kits
Other sources of materials that could be used as reference materials
JRC, FARRP
Effect of processing on extraction/kit performance
Most kits are not validated using model foods
International call for use of model foods spiking provides some useful
information but manufacturing gives best information about how a kit will
work

Food Allergy Research and Resource Program 2005

 Model Foods
Model foods must be made on a pilot plant or industrial-
sized scale
Ex. Simply making mini-cookies in a home-sized oven does not
mimic industrial practices
Results not practical or useful for the food industry
Invaluable for assessing how a kit is going to work with a specific
commodity and how efficient extraction method is
Becoming more important to use these types of standards in
assessing a kits performance for certain commodities/processing
Spiking is really pass in this area - only good for initial assessment
of possible matrix interferences

Food Allergy Research and Resource Program 2005

Factors Affecting Test Performance
Extraction method sufficient, recovery good?
Some foods are challenging (ex. tannins/polyphenols in
dark chocolate bind protein, high fat level hides allergen
in other types of ingredients)
Hydrolysis cannot analyze hydrolyzed or
fermented ingredients
Methods meant to detect intact proteins, not peptides
Processing

Food Allergy Research and Resource Program 2005

Factors Affecting Test Performance
Processing
Most kits for most allergens have good reactivity with
processed forms of allergenic food
Use of polyclonal antibodies and crude extracts, and making
antibodies against processed forms are recipes for successful kits
Monoclonals ok if against a heat-resistant epitope
Some of the egg residue kits have some issues in this
regard
Industry has been able to adjust and adapt many survey raw
material or use a kit that has antibodies against raw AND
processed egg

Food Allergy Research and Resource Program 2005

Factors Affecting Test Performance
Matrix effects
My lab has used all of the ELISA-based test kits available on the
market in our own validations and tests
Matrix effects not usually a problem for the vast majority, and kit
companies have added extraction additives to their extraction buffers to
assist in this regard, esp. with well-known matrix issues like dark
chocolate
Model foods of great use in assessing this; spiking useful also
Cross-reactivity
Even though most methods use polyclonal antibodies directed
against crude extracts, do not see cross-reactivity issues for the most
part

Food Allergy Research and Resource Program 2005

 Testing Issues
Hydrolyzed proteins
Most ELISAs are rendered useless when trying to
analyze for a hydrolyzed protein
But, a negative result does not mean that there is no
allergenic residue left -must ascertain residual
allergenicity via IgE methods (Western, RAST)
Another related area is analysis of fermented
ingredients (gums, Lactobacillus, etc.)
Companies do not tell contract labs the nature of their
samples, and when it comes out negative, they report
that to their customers!

Food Allergy Research and Resource Program 2005

 Food Testing Consumer
Reactions
My laboratory performs testing for food-
allergic consumers, their physicians, or
their lawyers (gratis) when they report a
reaction to a food
Food Allergy and Anaphylaxis Network, others
In ten years of doing this, we have only
seen large amounts of undeclared
allergenic food cause reactions

Food Allergy Research and Resource Program 2005

 Practical Issues for the Industry
Cannot currently do immediate monitoring
Technology does not exist lateral flow devices will help, but
not available for many allergens yet
Therefore, sanitation verification most practical, not test and
release
Do not have tests for some allergens
Ex. fish
Cannot test for hydrolyzed or fermented allergen
sources
Some types of cross-contact are not
homogenous or 100% cleaning is not possible
due to nature of product
Cannot take enough samples to practically test to be 100%
sure
In some of these cases, precautionary labeling justified, in
FARRPs opinion ex. dark chocolate and milk chocolate on
same line
Food Allergy Research and Resource Program 2005
Cross-Contact Associated with Milk-Allergic
Consumer Complaints
(Hefle and Lambrecht, J. Food Prot., Sept. 2004)
Product Casein (ppm)
Whole Wheat Roll 5,500
Tofu Cheesecake 16,100
Soy coffee drink 43,500
Dark Chocolate (kosher) 12,700*
Bakery icing (kosher) 44,500
Energy bar (dairy-free) 27,300
Cupcake (dairy-free) 13,700

Food Allergy Research and Resource Program 2005

 HRO (Highly Refined Oils)
What does HRO mean?
In FARRPs opinion, HRO = neutralized (alkali-refined), bleached, and
deodorized (NBD), or refined, bleached and deodorized (RBD)
Opinion based on scientific review of oral challenges with oils in the
literature
Available quantitative methods
Methods used in literature include ELISA, etc.
None of these have been validated in interlab trials or other types of
validation for protein-in-oil determination to date
Question as to whether small amount of protein in HRO is completely
extracted in aqueous buffer (usual method used), or whether some of the
more hydrophobic proteins stay in the oil fraction, and therefore not
extracted/determined
My lab uses an amino acid determination based on Edman degradation, but
also aqueous extraction (limitations), but report results as relative and not
a complete picture of the possible protein content of HRO oil
Food Allergy Research and Resource Program 2005
 HRO (Highly Refined Oils)
Protein levels of HRO reported in literature
Caveats aqueous buffers, epitope recognition by
antibody, etc., relating total nitrogen to intact
proteins, limitations of dye binding protein methods,
etc.
Protein levels reported in the literature
Usually a few mg/kg = few ppms

Food Allergy Research and Resource Program 2005