Protein based methods
Since allergens are, almost without exception, proteins, protein is the primary analyte that should be targeted. Protein based methods can be divided into two groups: Immunoanalytical methods and protein separation methods. Immunoanalytical (e.g. ELISA, RIE etc.) methods are mostly chosen because of the high specificity and sensitivity of the antibodies. They are used in food industry laboratories and by official food-control bodies to detect and quantify allergens present in food. Protein separation methods (e.g. MS) are based on the separation of proteins due to their variable size. They are mostly used to identify the allergenic protein.
ELISA
Enzyme-Linked Immunosorbent Assays (ELISAs) have been much favoured in allergen analysis. The specificity and sensitivity of ELISA technology, with limits of detection or quantification at low mg/kg level, make it a simple tool for allergen detection and quantification, allowing relatively fast and high throughput analysis. It is widely used in food industry laboratories and by official food-control bodies to detect and quantify allergens present in allergenic food or commodities. So far, ELISA test kits validated for defined matrices include peanut (in cereals, cookies, ice cream and chocolate; under the auspices of AOAC and EC JRC, Park et al 2005, Poms et al 2005) and hazelnut (in cereals, ice cream and chocolate; under the auspices of the German Federal Office for Consumer Protection and Food Safety, BVL).
Lateral Flow devices (LFD)
Related antibody-based technologies which are semi-quantitative include dipsticks and lateral flow devices (LFD) and are well suited to testing outside of the laboratory (e.g. monitoring clean-down of food processing lines), where a rapid result is required or qualitative results are needed for only a few samples.
Mass spectrometry (MS)
In the long term future it is possible that mass spectrometry methods will provide a viable alternative confirmatory method since they have the potential to detect protein (and therefore the hazard itself), provide information on sequence (giving species specificity comparable to DNA methods) and detect allergen contamination at low levels similar to those achieved by ELISA and PCR. The automated nature of mass spectrometry experiments and minimum of user involvement naturally lends itself to high-throughput quantitation. As with any new methodology its future application on analysis of food allergens is hampered by high equipment costs and the needs for specialist expertise in method development.
DNA-based methods
PCR / real time PCR
PCR can be a valuable tool to indicate presence / absence of an allergenic food or commodity if no suitable ELISA is available, if multiscreening of several allergenic foods is required or as a confirmatory analysis to ELISA (table 1). Whilst not measuring the actual hazard they have advantages in terms of high species specificity. Despite challenges such as extreme fragmentation of DNA or inhibition by remaining metal ions, lipids or proteins after filtration, indirect monitoring of allergen-containing products at low DNA level by PCR (below a concentration of 10 mg/kg) is possible.
Table 1: Comparison of protein-based and DNA-based allergen detection and quantification methods.
Since allergens are, almost without exception, proteins, protein is the primary analyte that should be targeted. Protein based methods can be divided into two groups: Immunoanalytical methods and protein separation methods. Immunoanalytical (e.g. ELISA, RIE etc.) methods are mostly chosen because of the high specificity and sensitivity of the antibodies. They are used in food industry laboratories and by official food-control bodies to detect and quantify allergens present in food. Protein separation methods (e.g. MS) are based on the separation of proteins due to their variable size. They are mostly used to identify the allergenic protein.
ELISA
Enzyme-Linked Immunosorbent Assays (ELISAs) have been much favoured in allergen analysis. The specificity and sensitivity of ELISA technology, with limits of detection or quantification at low mg/kg level, make it a simple tool for allergen detection and quantification, allowing relatively fast and high throughput analysis. It is widely used in food industry laboratories and by official food-control bodies to detect and quantify allergens present in allergenic food or commodities. So far, ELISA test kits validated for defined matrices include peanut (in cereals, cookies, ice cream and chocolate; under the auspices of AOAC and EC JRC, Park et al 2005, Poms et al 2005) and hazelnut (in cereals, ice cream and chocolate; under the auspices of the German Federal Office for Consumer Protection and Food Safety, BVL).
Lateral Flow devices (LFD)
Related antibody-based technologies which are semi-quantitative include dipsticks and lateral flow devices (LFD) and are well suited to testing outside of the laboratory (e.g. monitoring clean-down of food processing lines), where a rapid result is required or qualitative results are needed for only a few samples.
Mass spectrometry (MS)
In the long term future it is possible that mass spectrometry methods will provide a viable alternative confirmatory method since they have the potential to detect protein (and therefore the hazard itself), provide information on sequence (giving species specificity comparable to DNA methods) and detect allergen contamination at low levels similar to those achieved by ELISA and PCR. The automated nature of mass spectrometry experiments and minimum of user involvement naturally lends itself to high-throughput quantitation. As with any new methodology its future application on analysis of food allergens is hampered by high equipment costs and the needs for specialist expertise in method development.
DNA-based methods
PCR / real time PCR
PCR can be a valuable tool to indicate presence / absence of an allergenic food or commodity if no suitable ELISA is available, if multiscreening of several allergenic foods is required or as a confirmatory analysis to ELISA (table 1). Whilst not measuring the actual hazard they have advantages in terms of high species specificity. Despite challenges such as extreme fragmentation of DNA or inhibition by remaining metal ions, lipids or proteins after filtration, indirect monitoring of allergen-containing products at low DNA level by PCR (below a concentration of 10 mg/kg) is possible.
Table 1: Comparison of protein-based and DNA-based allergen detection and quantification methods.
| Protein-based methods (ELISA, Dipstick) |
DNA-based methods (real-time PCR, PCR-ELISA) |
|
| Detectability | Major allergen (group) or proteins specific for the offending food | DNA-fragment |
| Specifity | Cross reactions possible | Highly specific |
| Limit of detection | Low ppm range | Theoretically 10 molecules |
| Quantification | Quantification of specific protein | Quantification of copy numbers, calculating the protein content |
| Natural variability of target | Results may vary with species variety, climatic + seasonal changes | Genotype is very stable |
| Matrix effect | Minor changes in protocol can improve extraction | PCR inhibitors present in food are hard to avoid |
| Effects of food processing | Denatured or enzymatically modified proteins may not be detected | stable against high temperature, but DNA will be fragmented by low pH |
| Reference material | Not yet available | Not yet available |
| Sample preparation | Easy and fast | More laborious |
| Time required | 0.3 – 3.5 hours | 2 – 6 hours |
| Handling | simple | Training in DNA handling required |
| Stability of reagents | Several months at 4°C | Several years at -20°C |
| positive | intermediate | negative |
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