Endogenous allergen assessments - Europabio

Design, relevance and limitations of
current endogenous allergen
assessments
Scott McClain, Ph.D.
Syngenta Crop Protection, LLC
Research Triangle Park, NC USA

Design of studies is based, in part, on available
technology
2
-Serologydetection
of allergens
with patient IgE or
animal IgG antibody
-Protein Separation-
Visualize individual
proteins with
stains/serum IgE using
electrophoresis as a
foundation
-Analytical-
Mass spectrometry
is the newest
multiplexing,
quantitative
methodology

IgE, ug/ml
Question: Are Endogenous Allergens in Biotech Crops
Increased Relative to Their non-GM Counterparts?
● Has been applied to GM soybean because soybean is considered a
major allergenic food
● Traditionally, the technology available was based on serology:
3
- Use soybean allergic sera as a detection reagent for allergen-IgE
binding - measure allergen content.
• Total anti-soybean IgE binding ELISA – measures IgE that is specific
to “all” of the allergens a person may be responsive to; quantitatively,
if validated.
• Gel Electrophoresis and Western blotting; can observe binding to
multiple, individual allergens.
Total
IgE
To discriminate individual allergens by ELISA with
serum, individual ELISAs with protein standards
would need to be validated – for soybean this
would minimally be > 8 separate assays*.
Allergen #1
* - University of Nebraska, FARRP allergenonline.org

Protein Separation and Western Blotting
● Western blotting (WB), and in particular 2-dimensional WB, is of
interest for observing individual proteins
4
- A qualitative observation of the proteins binding serum IgE antibody.
• Although “spots” can be optically measured in general, the
technique is considered semi-quantitative since there are
no standard curves assessed for individual allergens (in
most cases).
- Also perceived as beneficial for observing “new” allergens that
may be present in GM variety…
this is improbable given that new varieties are assessed
prior to market release and exposure. There is also no
premise for new proteins, much less new allergens being
created by introduction of GM trait(s).

2-D Electrophoresis and Western Blotting
Total protein stain shown – Red circle indicates observable soybean proteins by Western
5
Gel Electrophoretic pattern
Taken from Batista et al., 2007. Int Arch Allergy Immunol 2007;144:29–38
IgE reactivity to all of the
possible soybean
allergens is very limited
in a single serum

Considerations for Serology Based Studies
6
Primary Limitation – limited availability of well-characterized sera.
- Variability in IgE binding across one or more proteins among different
patients.
• Not all patients have IgE against a given allergen of the same
affinity – thus, quantitative determinations are limited.
- Soybean has many allergens, so thoroughly characterizing the exact
same set of proteins with good reproducibility from one study to the
next is not possible since sera will vary temporally among patients.
- Virtually impossible to have same set of patient sera form one study to
next

Liquid Chromatography MS/MS for Protein
Identification
7
1. Proteins are digested into peptides
2. Peptides are separated to reduce complexity
3. Peptides are “weighed” and sequenced by mass spectrometry
4. Peptide sequence provides protein identification through bioinformatics
S.E. Stevenson, Houston N., and Thelen J. 2010. Regul. Toxicol. Pharmacol.
58, 3, S36-41.

AQUA® Peptide Analysis by MS to Quantify Proteins
Two different
isotopes to label
amino acids
These labeled
(heavier) peptides
can then act as
peptide
Standards
Isotope labeled
Native peptide
AQUA peptide
8
H 2N
H 2N
HO
C
NH
CH 2
CH 2
CH 2
CH
C O
NH
Arginine (R) – 174.2
13 C6H 14 15 N4O 2 – 184.2
1 2
H 2N
HO
NH 2
CH 2
CH 2
CH 2
CH 2
CH
C O
Lysine (K) – 146.19
13 C6H 14 15 N2O 2 – 154.19
LSAEFGSLR – 978.51 VSDDEFNNYK – 1229.52
LSAEFGSLR – 988.51 VSDDEFNNYK – 1237.52

Aqua® Peptides and Quantifying; How does it work?
9
Seed allergen peptide (previously identified)
signal
Mix label
with seed
prep
mass
Labeled peptide standard
+
mass spectrometry can distinguish them
+
+
Known amount of
standard added
Ratio gives quantity

Relevance of the Study Design for Endogenous
Allergen Content
● Premise:
10
- Endogenous allergens could, by some unknown mechanism, be
influenced to express at a higher level in GM-traited soybean crops
compared with non-GM
- Soybean – although soybean has many allergens, including
abundant seed storage proteins, the threshold exposure to individual
allergens is not known.
• Therefore, at this point in time, there is no knowledge of how
many µg or mg of an allergen is significant from an allergy safety
perspective –
• this in turn limits the study design - a single comparison of GM
versus non-GM control is not useful
- The only relevant study that can be designed is to survey several
non-GM references so there is context to the study in terms of the
natural range of expression for one or more allergens.

µg allergen protein/mg total protein
Example – Observing Allergens in the Context of
a Population of Non-GM Varieties (References)
11
120
100
80
60
40
The difference is small
relative to the variability
in the references
GlyG1
GlyG2
Allergen
Test
Control
upper TI
lower TI
Many references can be used to
show the probable range of
expression for a population
The differences between the GM
“test” and its non-GM” control can
then be assessed against the natural
variability in allergen expression
Conclusion – in this case, both
allergens show small differences for
the GM variety that are not
biologically relevant

Allergen Context: Understanding Natural Variability
12
Many non-GM reference samples help establish
the concentration range for a new protein (i.e.,
allergen) that has clinical implications; particularly
when employing quantitative approaches
Why is this important ?
ug of allergen per mg protein
If a protein is being characterized
for the first time, it is critical to
validate the population for its
“normal” content
In the future – the quantity of an
allergen could potentially be
correlated to threshold exposure
levels to understand allergy
impact

Soybeans
13
Glycine max
Yes, it’s an
allergenic food
But,
We don’t know how much or
how little causes allergy for
any one of the many protein
allergens
And,
We don’t have evidence that
GM varieties would be
expected to differ in their
allergen content
For now,
We can assess GM and
non-GM, but not in
isolation
Small differences are
unlikely to be relevant to
allergy risk – Therefore,
we need to understand
small differences as best
as we can
We cannot predict endogenous
allergy risk relative to a
measured amount of protein…
So, any assessment has to
include a population of
references to understand the
natural variability for any single
allergen

Endogenous Allergen Approaches
● Question: Are Endogenous Allergens in Biotech Crops
Increased Relative to Their non-GM Counterparts?
● Question implies that the target protein(s) can be quantitatively
measured with relevance to allergy risk
14
- ILSI/HESI protein allergenicity tech committee has
supported work to use quantitative approaches to provide
measures of soybean allergens
● Methods may include qualitative or quantitative
approaches
● Methods should be:
• Sensitive
• Specific
• Reproducible

Relevance of the data – Can this data be
interpreted for allergy risk?
● Can we measure allergens?
15
- Yes, ability is increasing all the time and with increasing sensitivity
● What is the relevance to the population in terms of their risk for allergy?
- Most allergens still require further study to characterize their
sensitization potential and the amount that causes clinical allergy
- There is no existing hypothesis for a change to endogenous allergy
risk based on GM varieties
● So, even though we have the ability to measure allergen content the
question remains – have we added value to the allergenicity
assessment?

Contributing EuropaBio Member Companies
● Bayer CropScience
● BASF
● Dow AgroSciences
● Dupont Pioneer
● Monsanto
● Syngenta
16

Questions and Discussion
● Thank your for your attention
17