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STRONG QC & QAQA
STRONG QC &

Beyond Immuno-Based

Allergen Testing

Most commercially available kits for food allergen testing Furthermore, PCR can be used for allergens like celery which
rely on the application of immuno-based methods such cannot be detected by antibodies. Celery has to be labeled in the
EU but until now, all attempts to produce reliable antibodies have
as ELISA or lateral flow devices (strip tests). To carry failed due to the close relationship between celery and other plants
out ELISA, trained personnel are required but numerous like parsley, carrot, coriander or fennel.
samples can be analyzed in parallel by using 48-well or Mass Spectrometry: A High-end Technology
96-well microtiter plates. In general, the analysis can take An even newer technology for detecting and quantifying allergens
between 30 minutes and a few hours. is mass spectrometry, a high-tech method that identifies proteins
and peptides with a very high level of accuracy. The first attempts at
applying this technology to allergen detection began in the late 1990s
At present, ELISA is the most widely applied method for the detection but most of the results were only published in the last few years.
and quantification of food allergens. However, although many samples The main benefit of using this technology for allergen testing is
can be analyzed at the same time, these samples can only be tested for the high level of confidence and reliability. The instruments can detect
one analyte.
multiple peptides per protein. Ideally, two to three fragment peptides
Limitations to Consider are analyzed per allergen.
The advantage of this approach is that even if proteins are partially
Due to the high specificity of antibodies towards only one particular degraded or modified due to harsh food processing conditions, the
allergenic protein and technology-related limitations, a separate kit has to probability of finding at least one intact fragment is quite high. These
be used for each allergen. Furthermore, the high degree of specificity to marker peptides are selected from databases or from literature and
one allergen might lead to false negative results. Food processing steps must be highly specific for the allergens to be quantified. Furthermore,
like heat treatment, the addition of acidic compounds or fermentation can they are chosen to be resistant to food processing alterations.
modify the target protein structure. These modified allergens can lose This multi-peptide recognition strategy of the allergen is not
their immunological properties and the antibody – target protein complex possible with immuno-based assays. Antibodies usually bind to only
cannot be formed anymore. This leads to false negative results or reduced one particular (immunogenic) fragment of the allergen. If this small
quantifications. fragment is modified, the recognition of the target might be hampered.
Strip tests are inexpensive, very easy to use, do not require laboratory Additionally, mass spectrometry is able to measure several allergens
equipment, and give results usually in a few minutes. However, most strip in parallel. These multi-analyte methods have become particularly
tests are only qualitative and rely on antibodies as recognition elements.
popular in recent years. This innovative strategy allows a single
Detecting Allergens with DNA extraction of a sample to be screened for numerous allergens in a
single analysis run.
PCR (polymerase chain reaction) is a relatively fast and inexpensive
method for identifying DNA. This technology, developed in the 1980s, has No One-size-fits-all
improved continuously since then. PCR has been used for many years The perfect method, a gold standard for allergen quantification, does
in the fields of medical diagnostics, forensics, environmental monitoring, not exist. ELISA and LFDs are the method of choice for the majority
and the quantification of genetically modified organisms in food and feed. of industrial applications. Results can be obtained relatively quickly,
In the early 2000s PCR was applied for the first time to identify the costs are moderate to low and personnel can be easily trained to
DNA of common food allergens like hazelnut and peanut. Until now, PCR use these tests. For some problems like highly processed testing
assays for most of the US “big eight” and the 14 EU food allergens have material or specific analytes, PCR might lead to better results. Mass
been published. spectrometry is situated at the upper end of available technologies but
The fact that PCR detects the extremely stable DNA molecule might is still in its infancy for allergen testing. However, it has, in recent years,
be an advantage when analyzing highly processed food. DNA tends to be become the method of choice for many other analytical challenges.
unaffected even by extreme conditions and can therefore still be detected It can be expected that this technology might experience a boost in
even when most of the proteins have already been degraded or modified the field of allergen analysis in the near future.Figure 1 A simplified
in some way.
scheme of PCR analysis

52 FOOD FOCUS THAILAND JUL 2018