Allergen testing

[rajendran]

Allergic reactions to foods can be provoked by less than 1/5000th of an ounce of an allergen. Such trace amounts are well above the levels caused by cross-contamination in poorly developed industrial food manufacturing. Another factor is that allergens can be disguised in food ingredients. For example, soy lecithin obviously comes from soybeans (soybeans are one of the so-called 'big eight' allergens), but less obvious is that lecithin not made from soy likely contains egg (another 'big eight' allergen). But under existing labeling laws, it is not required for soy or egg to be specifically identified on a food label of a product containing one of these ingredients, making it confusing or difficult for someone with food allergies to make an informed decision. Millions of North Americans suffer from food allergies. Some food allergies, particularly peanut allergies, can be fatal. Eight food groups are responsible for most allergic reactions: Crustaceans such as crab and lobster; peanuts, eggs, fish, milk, soy, tree nuts such as almonds and walnuts; and wheat.

REGULATION
On August 2, 2004, the Food Allergen Labeling and Consumer Protection Act (S. 741) in USA was signed into law. The bill requires food manufacturers to clearly state if a product contains any of the eight major food allergens responsible for 90% of all allergic reactions: milk, eggs, peanuts, tree nuts, fish, shellfish, wheat, and soy. In addition, it requires that the Food and Drug Administration conduct inspections and issue a report within 18 months to ensure that food manufacturers comply with practices to reduce or eliminate cross-contact of a food with any major food allergens that are not intentional ingredients of the food.
The use of protein additives such as gluten, peanut and milk proteins is widespread. They add to the functionality, texture and taste of many foods. However, for an increasing number of people (perhaps 6-8% of children and 2% of adults), these and other proteins can cause serious problems of intolerance and allergenicity. Addition of these ingredients to foods either accidentally or without adequate labelling can only be controlled if effective methods of detection are employed by food manufacturers and regulatory laboratories.

TESTING
ILC Micro-Chem can facilitate the detection of an increasing number of these allergens in foods, using laboratory ELISA or 'on-site' Rapid Tests that employ lateral flow methods. The BioKits Allergen Swabbing Kit has been designed to provide an effective swabbing method and sample collection device. The potential allergen containing solution produced can be tested immediately (using BioKits Rapid Tests) or simply send the samples to our laboratory for analysis. These specific allergen tests supplement your regular hygiene monitoring tests.

SWABBING
A list of potentially allergenic ingredients* has been defined. Control of these allergens in e.g. food manufacturing & catering establishments, is therefore becoming increasingly important. Many manufacturers are adapting HACCP programs to include an Allergen Control Plan to minimize risks to allergic consumers. High levels of cleanliness are essential to prevent Cross-contamination of all process equipment.

(*including Cereal gluten, Crustacea, Fish, Egg, Milk, Peanut, Sesame, Soya, Tree nuts)

The CFIA recognizes the efforts by many members of the food industry to improve the accuracy of ingredient declarations and to implement controls to reduce carry-over of ingredients. As food safety is paramount to consumers, the food industry, and government, the CFIA also urges the food industry sector to develop strategies, such as an allergen prevention plan, to manage the risks associated with those foods known to cause severe adverse reactions. part of every strategy should include a thorough evaluation of the manufacturing and ingredient control procedures. It is also the importers/distributors responsibility to ensure that all prepackaged foods that are imported are fully and correctly labelled, and preferably are sourced from suppliers having an allergy prevention plan in place.

Accurate and complete labelling of foods will reduce the need for costly food recalls. It will also assist Canadians with severe food sensitivities to make safe choices from a wider variety of foods in the marketplace.
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for more details visit the website given below
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ilcmicro-chem.com/module/allergen-testing.php

[Zagami]

Major apple allergen Mal d 1 extract
Native molecule : apples were purchased at the local market, and 100 g of fresh unpeeled apple without core and stalks was homogenized in a blender at -4 °C in 200 ml of pre-cooled (-20 °C) diacetone alcohol (4-hydroxy-4-methyl-2-pentanone) for 10 min. The homogenate was mixed with 300 ml of pre-cooled acetone and equilibrated overnight at -20 °C. After removal of the supernatant by filtration through a glass fiber filter, the precipitate was washed twice with pre-cooled acetone and once with pre-cooled acetone:ether (1:1, v/v) and then air dried. The protein extract was resolubilized in 200 ml of PBS contained 2.0 mM ethylenediaminetetraacetic acid (EDTA), 5.0 mM sodium diethylthiocarbamate, 0.5 mM benzamidine hydrochloride, and 0.2 mM phenyl methyl sulfonyl fluoride, by stirring for 1 h at 4 °C. After centrifugation for 45 min. at 40,000 g and 4 °C, the extract was lyophilized and stored at -20 °C. Before to use resuspend lyophilized in few ml of dH2O and remove additives by extensive dialysis against distilled water for 24 h. The total amount of protein was quantified by the micro BCA Protein Assay.

[Zagami]

Pathogenesis-related proteins (PRPs) also called "plant defense-related proteins" or "stress-inducible plant proteins" has been classified into 14 classes some of which (classes 2, 3, 4, 5, 9, 10, 14) are richer in substances with allergenic properties - together with other classes of proteins (alpha amylases and proteases inhibitors) they form the "plant defence system". Vegetables produce PRPs in response to infection or after plant injury or application of chemicals : long-term conservation and methods used for rapid artificial ripening of vegetables can cause plant to produce PRPs or other allergens. Therefore the correspondence between the allergen content of a vegetable, positive skin tests and the presence of clinical symptoms is a highly complex matter that depends on the environmental conditions under which the vegetal was grown, stored, and processed as a foodstuff. Today doesn't exist diagnostic tests vivo/vitro for food allergy-intolerance with sufficient sensitivity. Yet even in the most clear-cut cases, that is, patients with positive histories and positive prick tests, it actually does so in no more than half the cases. In the other 50% of subjects with a clear-cut history and in all those whose history is less clear or who have negative prick tests. The chief reason of this failed reaction is that natural extracts of plant proteins are extremely labile. Hence diagnostic procedures in vivo and in vitro often yield irreproducible results.

[Zagami]

Nasal Eosinophil Peroxidase (NEP)
Nasal mucous were diluted 1:10 in Hepes buffer 50 mM at pH 8.0 (dilution buffer NEP). Dispense in MTP BioHit 75 µl of diluite sample in quadruplicate and then dispense in 2 wells 150 µl of cold stop solution (background), see above, and dispense in all wells, comprising background wells, 75 µl of substrate (Hepes buffer 50 mM at pH 8.0, 6 mM KBr, 3 mM o-phenylenediamine [OPD], 8.8 mM H2O2) and incubate for 30 sec at room temperature. Dispensing 150 µl of cold stop solution (4 N H2SO4, containing 2 mM resorcinol). Read l’Abs at 490 nm. As negative control dispense 75 µl of dilution buffer, 75 µl of substrate and, after 30 sec., stopped reaction dispensing 150 µl of cold stop solution. The enzyme activity of the nasal mucous samples were calculated by subtracting the mean background OD and are expressed as change in optical density per minute.

[Zagami]

Nasal Neutrophil myeloperoxidase (NMPO)
Nasal mucous were diluted 1:10 in citrate buffer 10 mM at pH 5.0 (dilution buffer NMPO). Dispense in MTP BioHit 75 µl of diluite sample in quadruplicate and then dispense 75 µl of substrate (3 mM of 3,3,5,5-tetramethylbenzidene dihydrochloride [TMB], 120 mM resorcinol, and 2.2 mM H2O2 in dH2O) and incubate for 2 min at room temperature. Stopping reaction dispensing 150 µl of cold stop solution (4 N H2SO4). Read l’Abs at 450 nm. As negative control dispense 75 µl of dilution buffer, 75 µl of substrate and, after 2.0 min., stopped reaction dispensing 150 µl of cold stop solution. The enzyme activity of the nasal mucous samples were calculated by subtracting the mean background OD and are expressed as change in optical density per minute.