Food Biochemistry and Food Processing (2 edition)

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810 Part 8: Food Safety and Food Allegens

Another area of interest is specific oral tolerance induction

(SOTI). Taking milk as an example, about 15% of children with

milk allergy maintain the susceptibility permanently in their

life. Strict avoidance of cow’s milk and cow’s milk derivatives

remains the gold standard for allergen management for such

CMA patients. For these patients, however, the likelihood of

exposing the offending allergen unintentionally always remains

and thus total avoidance cannot be guaranteed. Similar concerns

exist for patients with persistent peanut and tree nut allergies.

SOTI is a promising approach particularly for patients with per-

sistent food allergy. Staden et al. (2007) reported that SOTI

treatment (using a daily dose of CMP, starting from 0.002 mg

CMP) remarkably increased the threshold dose for allergic reac-

tion in CMA patients. As a result, the patient could be protected

against potentially fatal allergic reactions when small amounts

of the hidden allergen are accidentally ingested. Since this ap-

proach uses the specific offending allergen to induce tolerance,

development of mild to moderate side effects (such as itching

in the mouth, nausea and wheals, etc.) is very common. Close

supervision by an experienced professional is, therefore, neces-

sary for SOTI treatment. Similar promising studies have been

reported for peanut (Blumchen et al. 2008, Clark et al. 2009,

Jones et al. 2009).

For the food industry, food allergen testing has become an

increasingly important tool for the management and control of

allergens during food production and processing. The rest of

the chapter provides a brief summary of some of the methods

currently available for allergen detection.

METHODS FOR DETECTING ALLERGENS

Table 42.4 provides examples of some of the commonly used

methods for food allergen detection. Food allergens may be

detected either by the presence of the target allergen itself or

by using a marker in the target food. The method of choice

will depend on factors such as food matrix interference, nature

and quantity of the target allergen, the desired level of detection,

specificity and time and resources required for running the assay.

The threshold dose for sensitised individuals to manifest allergic

reactions is quite low and variable among individuals (Bindslev-

Jensen et al. 2002). However, it is commonly agreed that food

allergen detection methods should be sensitive enough to detect

1–100 mg of analyte (allergen) per kg of processed food (Poms

et al. 2004). Increasing numbers of rapid and user-friendly test

kits to detect different food allergens are now commercially

available with sensitivities ranging from 0.05 to 10 mg/kg (Table

42.5).

Protein-based food allergen detection methods include im-

munoblotting, rocket immunoelectrophoresis, enzyme aller-

gosorbent test, enzyme-linked immunosorbent assay (ELISA),

protein microarray and biosensors. More recently developed

DNA-based methods are available as supplementary and com-

plementary methods to protein-based methods and are particu-

larly useful in species differentiation and detection of genetically

modified food (Mustorp et al. 2008). This chapter will focus on

ELISA-based detection, lateral-flow assays (LFA) and dipstick

test, proteomic approaches and DNA-based allergen detection

methods.

ELISA-Based Detection Methods

Among the growing number of food allergen detection tech-

niques, ELISA has gained prominence and has become an exten-

sively used technique due to its high sensitivity and specificity,

availability of automation and user friendliness. Some ELISAs

are designed to detect specific allergens (e.g. BLG, Ara h1 (a

major peanut allergen) and shrimp tropomyosin), whereas other

ELISAs detect mixtures of proteins from the allergenic source

(e.g. total milk, egg, peanut and almond-soluble proteins; Taylor

et al. 2009). Detection is based on binding of an allergen or a

specific marker protein with an antibody specifically generated

to recognise and bind to these proteins. This binding complex

is visualised by a colourimetric assay when the enzymes, which

are labelled to the allergen specific antibody, interact with the

substrate solution. The concentration of the allergen can be quan-

tified by obtaining optical density (OD) values with a microplate

reader and plotting these values using a standard curve.

There are certain limitations of ELISA-based detection meth-

ods, however (Yeung 2006). ELISAs are based on aqueous sys-

tems and they do not work well in detecting insoluble proteins or

allergens derived from edible oil-producing foods. In addition,

ELISA may not detect oleosins, a family of protein involved in

the formation of oil bodies in peanut and sesame (Pons et al. 2002

and Leduc et al. 2006) and soya lecithin containing more than

50 ppm residual protein (Taylor et al. 2009). ELISAs also fail

to distinguish certain closely related foods. For example, walnut

antisera react with pecan (Niemann et al. 2009) and mustard an-

tisera react to rapeseed (Lee et al. 2008). These may be caused by

the presence of cross-reactive epitopes or proteins between two

foods (Taylor et al. 2009). Regardless of the above-mentioned

limitations, ELISA is still a very powerful tool and remains

the method of choice for food allergen detection. ELISA-based

food allergen detection methods are developed using either the

sandwich ELISA or competitive ELISA format.

Sandwich ELISA

In this assay, as indicated by its self-explanatory name, an al-

lergen of interest is captured between two allergen-specific an-

tibodies. The first allergen-specific antibody is immobilised on

the solid phase to capture the allergen, and the second allergen-

specific antibody is labelled with enzyme to detect the captured

allergen. Allergens need to have enough binding sites, for ex-

ample, large molecules such as proteins, to allow binding of the

two allergen-specific antibodies (Schubert-Ullrich et al. 2009).

Competitive ELISA

This assay is based on the competitive binding of unknown ana-

lyte (allergen) in a sample of interest and a known analyte (aller-

gen) to the allergen-specific antibody. The antigen of interest in

this case is incubated in the presence of the unlabelled antibody.

Antibody/antigen complexes are formed and the solution is then