1. Development of a Multiplex Immunoassay
for Food Allergen Proteins
Ross Yarham, Anna Kuklinska-Pijanka MSc, David Gillick, Elizabeth Young,
Martin D. Chapman PhD, James P. Hindley PhD
Indoor Biotechnologies, Ltd., Cardiff, Wales, UK
Ross@indoorbiotech.co.uk; www.inbio.com
In order to help allergic patients manage often severe symptoms, food manufacturers are required to list allergens on their products and researchers are
working to develop effective immunotherapies. Due to limitations of the existing tools, precise identification of the allergens in foods, therapeutic and
diagnostic products can be difficult.
Aim: We sought to develop an accurate, sensitive and reliable assay that would enable simultaneous quantification of multiple food allergens.
INTRODUCTION
Using microsphere-based technology (Luminex Corp., Austin TX, USA) we developed a ‘proof-of-concept’ multiplex
immunoassay that enables simultaneous quantification of major food allergens from peanut (Ara h 1, Ara h 2 and
Ara h 6), milk (Bos d 5) and egg (Gal d 1).
Fluorescent microspheres (beads) were coupled to allergen specific monoclonal antibodies (Figure 1.). Detection of
the target allergens was accomplished using biotinylated specific mono- or polyclonal antibodies (Table 1.). and
streptavidin conjugated fluorochrome. Highly purified natural allergens (Figure 2.) were used to generate a
standard curve for each protein (Figure 3.). Allergen content was measured in various sample types including
peanut flour, milk powder, egg powder and NIST peanut butter. The results were compared to ELISA.
MATERIALS AND METHODS
RESULTS
• A ‘proof-of-concept’ multiplex immunoassay for simultaneous quantification of major food allergens has been developed.
• The multiplex immunoassay requires much smaller samples than ELISA.
• The ‘open-architecture’ multiplex platform allows for addition of further allergens to the list of analytes and creation of a wider ‘food-panel’.
• The array can provide a robust, rapid and cost effective alternative to existing methods for research, pharmaceutical, biotechnology and food industries.
CONCLUSIONS
Table 2. Performance characteristics of the food multiplex assay (*Mean CV% of average allergen concentration for triplicate samples run on the same
plate; ** Mean CV% of average allergen concentration for samples analysed on three separate days; *** Mean CV% of average allergen concentration
for at least 3 serial dilutions).
Target food Target protein Standard Capture Antibody
Detection
Antibody
Peanut
Ara h 1 nAra h 1 mAb 2C12 mAb 2F7
Ara h 2 nAra h 2 mAb 1C4 pAB
Ara h 6 nAra h 6 mAb 3B8 mAb 3E12
Milk Bos d 5 Bos d 5 mAb 97N mAb 117N
Egg Gal d 1 LoTox nGal d 1 mAb CB-2 mAb CB-1
Table 1. Proteins and antibody pairs used for development of the food multiplex assay.
Figure 2. SDS-PAGE of purified natural Gal d 1, Bos d
5, Ara h 1, Ara h 2 and Ara h 6 used as standards in
food multiplex assay (Purified using affinity
chromatography or multistep chromatography).
1.
4.
2.
3.
5.
Figure 1. 1. Fluorescent microsphere; 2. Allergen
specific antibody coupled to the bead; 3. Target
protein; 4. Allergen specific biotinylated detection
antibody; 5. Streptavidin-PE.
Disclosure: In relation to this poster I declare the following, real or perceived conflicts of interest: All authors are employees of Indoor Biotechnologies.
REFERENCES
Earle CD, King EM, Tsay A, Pittman K, Saric B, Vailes L, Godbout R, Oliver KG, Chapman` MD. High-
throughput fluorescent multiplex array for indoor allergen exposure assessment. J Allergy Clin Immunol.
2007;119:428-33
King EM et al.. Simultaneous detection of total and allergen-specific IgE by using purified allergens in a
fluorescent multiplex array. JACI 2007; 120:1126-31
We would like to thank Eva King, Stephanie Filep, Sabina Wünshmann, Bryan Smith, Anna Pomes and
Denise Block for support in developing the assay.
The work was partly funded by iFAAM project (EU FP7, Integrated Approaches to Food Allergen and Allergy
Risk Management)
ACKNOWLEDGEMENTS
• The food multiplex assay was able to measure
multiple allergens in a small (<50µl), single
sample.
• Lower limit of detection (LLOD) of the assay was
as low as 30pg/ml for Ara h 6 (Table 2.).
• Sensitivity of the multiplex assay was increased
by up to 39-fold compared to ELISA.
• The multiplex food array produced reproducible
results showing intra-assay CVs<12% and inter-
assay CVs<16%.
• Results demonstrated good parallelism
(CVs<17%).
• Standard curves range between 5000-2.44
ng/ml for Ara h 1, 100-0.05 ng/ml for Ara h 2, 50-
0.02 ng/ml for Ara h 6, 200-0.1 ng/ml for Bos d
5, 3400-1.7 ng/ml for Gal d 1 (Figure 3.).
• Preliminary tests show correlation (R2) between
the peanut multiplex assay and ELISA of 0.90,
0.90, 1.00, 0.94 and 0.80 for Ara h 1, Ara h 2,
Ara h 6, Bos d 5 and Gal d 1 respectively
(Figure 4.).
Figure 4. Correlation between results obtained using ELISA and
multiplex food assay. Analyzed samples were: peanut flour extract,
NIST SRM peanut butter, diagnostic extract, milk powder, egg
powder, research chocolate dessert and cookie.
ELISA LLOD
(ng/ml)
Multiplex LLOD
(ng/ml)
LLOD fold
change
Multiplex
intra-assay CV%*
Multiplex
inter -assay CV%**
Multiplex assay
parallelism***
Ara h 1 31.5 2.5 12.6 2.2 15.6 16.2
Ara h 2 2.0 0.2 10 2.0 9.2 13.3
Ara h 6 0.8 0.03 26.7 4.7 14.4 11.2
Bos d 5 7.8 0.2 39 11.6 14.3 13.0
Gal d 1 6.6 5.8 1.1 4.1 2.7 8.6
Figure 3. Standard curves for Ara h 1, Ara h 2, Ara h 6, Gal d 1,
Bos d 5 in a food multiplex assay.