Food Biochemistry and Food Processing (2 edition)

BLBS102-c42 BLBS102-Simpson March 21, 2012 14:27 Trim: 276mm X 219mm Printer Name: Yet to Come

42 Food Allergens 813

modifications and more (Monaci et al. 2006). Many technolo-

gies, including protein separation via two-dimensional elec-

trophoresis or liquid chromatography (LC), mass spectrometry

and arrays (to map protein–protein interactions), are applied in

proteomic-based studies. Shefcheck and Musser (2004) showed

useful application of LC-based proteins separation with MS

analysis (LC-MS/MS) to detect peanut allergen Ara h1 in ice

cream with a detection level of 10 mg/kg. Proteomic studies can

offer more detailed information about allergens (Shefcheck and

Musser 2004), help in the identification of new allergens (Yu

et al. 2003) and provide objective evaluation of changes induced

by genetic modification (Herman et al. 2003). However, the

requirement of a highly skilled operator, the high cost of mass

spectrometers, the length of time required for sample preparation

and analysis, and the need for separate protocols and optimisa-

tion for each sample prevent current proteomic approaches to be

used for routine detection of food allergens. Nevertheless, pro-

teomics remains a promising tool in allergen studies due to its

great potential for detection and identification of food allergens

in complex mixtures. Recent developments in mass spectrometer

instrumentation and software offer ultra-high throughput, high

sensitivity, and high-resolution capabilities (Chassaigne et al.

2009). Currently, attempts to couple more efficient protein sepa-

ration (microfluidic) and detection technologies with MS analy-

sis are progressing. Proteomic approaches could therefore soon

be widely applicable for detection and identification of food

allergens.

DNA-Based Allergen Detection Methods

Detection of allergens in food, by traditional protein-based de-

tection methods, can sometimes be very difficult, as they are of-

ten present in minute amounts and are masked by the food matrix

(Keck-Gassenmeier et al. 1999). DNA-based allergen detection

methods have emerged as an alternative and complementary

method to detect allergens where effective protein-based aller-

gen detection methods are unavailable. DNA-based tests also

possess high accuracy and sensitivity and, in general, are able to

withstand harsh processing conditions better than corresponding

allergenic proteins (Mustorp et al. 2008). On the other hand, in

some food, DNA are partially or totally degraded and conse-

quently false-negative results could occur, while the food may

still contain the allergen. In addition, some food matrix com-

ponents may reduce DNA amplification efficiency. DNA-based

methods, in comparison to ELISA-based methods, are relatively

more complicated, require more equipment and have lengthier

protocols. For this reason, DNA-based methods have been con-

sidered impractical for routine detection. They may, however,

be desirable for use by regulatory agencies and in institutions

where there is easy access to appropriate equipment.

The sensitivity of the DNA-based methods strongly depends

on the amount and on the quality of the template DNA since the

DNA encoding the allergen in the food samples may be present

in low amounts and may also be degraded as a result of process-

ing (Holzhauser et al. 2006). Three DNA-based approaches are

currently available to detect food allergens.

Polymerase Chain Reaction (PCR) with

Gel Electrophoresis

The assay involves DNA extraction from food, amplification of

specific DNA fragment using primers that bind selectively to

complementary parts of the DNA to be amplified, followed by

agarose gel electrophoresis (Besler 2001). PCR consists of three

steps, determined by different temperatures in every cycle of

amplification: denaturation of double strands DNA, annealing

of the primers and extension of the primers by the enzyme Taq

polymerase (Poms et al. 2004). The amplified product is loaded

to run agarose gel electrophoresis and visualised by staining the

gel. PCR with gel electrophoresis approach is good for qualita-

tive detection of allergen in the food.

PCR with ELISA

This assay is a combination of a highly specific DNA-based

method with the relatively simple and highly sensitive ELISA

assay for semi-quantitative analysis (Poms et al. 2004). As in

other PCR-based methods, extraction and purification of DNA

from a food sample and amplification of specific DNA fragment

encoding to suspected allergen using primers in a thermocy-

cler are prerequisite for PCR-ELISA assay. The ELISA compo-

nent of the test involves immobilisation of the amplified PCR

product on a solid phase (a microplate), denaturation of double

strand, hybridisation with specific probe, detection of the probe

with enzyme-conjugated antibody and observation of the en-

zymatic colour reaction after addition of substrate (Holzhauser

et al. 2002). The DNA concentration can be determined via

absorbance obtained from an enzyme–substrate reaction.

Real-Time PCR

Real-time PCR allows gel-free detection ‘in real time’ with high

accuracy by using a target-specific oligonucleotide probe with a

reporter dye and a quencher dye attached (Desjardin et al. 1998).

Oliver and Vieths (2004) reported real-time PCR method to be as

sensitive as sandwich ELISA in detecting peanut allergens in 33

food samples. Many different types of real-time PCR assay kits

are commercially available to detect many of the priority food

allergens (Poms et al. 2004), including kits for detecting allergen

encoding genes from mustard, sesame and celery (Mustorp et al.

2008).

CONCLUSION

Food allergy will likely continue to be an important food safety

issue in the coming decades. Although substantial progress has

been made in the field in the last 20 years, much still remains

unknown. One area still requiring significant work is the ques-

tion of the minimum eliciting dose required to provoke reactions

in sensitised individuals. Increasing prevalence rates along with

the possibility of severe allergic reactions after exposure to very

low level of allergens in sensitised consumers continue to be

a concern. Although exact threshold doses for food allergens

are not known at present, levels of detection offered by current