Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum

Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum (http://www.molecularstation.com/forum/)
-   Chemistry Forum (http://www.molecularstation.com/forum/chemistry-forum/)
-   -   Allergen testing (http://www.molecularstation.com/forum/chemistry-forum/86278-allergen-testing.html)

rajendran 08-22-2012 03:35 PM

Allergen testing
 
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.
-------------------------------------------
for more details visit the website given below
--------------------------------------------
ilcmicro-chem.com/module/allergen-testing.php

Zagami 03-26-2013 02:29 PM

Re: Allergen testing
 
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 04-29-2013 11:51 AM

Re: Allergen testing
 
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 05-06-2013 01:46 PM

Re: Allergen testing
 
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 l of diluite sample in quadruplicate and then dispense in 2 wells 150 l of cold stop solution (background), see above, and dispense in all wells, comprising background wells, 75 l 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 l of cold stop solution (4 N H2SO4, containing 2 mM resorcinol). Read l’Abs at 490 nm. As negative control dispense 75 l of dilution buffer, 75 l of substrate and, after 30 sec., stopped reaction dispensing 150 l 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 05-06-2013 01:47 PM

Re: Allergen testing
 
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 l of diluite sample in quadruplicate and then dispense 75 l 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 l of cold stop solution (4 N H2SO4). Read l’Abs at 450 nm. As negative control dispense 75 l of dilution buffer, 75 l of substrate and, after 2.0 min., stopped reaction dispensing 150 l 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 01-07-2014 07:35 PM

Re: Allergen testing
 
Useful of total IgE determination in allergic diseases
Measurement of total IgE, in serum, secretion or on cell surfaces is of little diagnostic value. The reason is that mitogenic factors in viruses (e.g., Cytomegalovirus - CMV), bacteria (e.g., Staphylococcus), helminths (e.g., Ascaris, Schistosoma) and adjuvant factors in air pollution (e.g., cigarette smoke, and diesel exhaust) stimulate the production of IgE molecules without initiating any allergen specific IgE-sensitization. For the last issue it has been theorised that smokers may have a higher than average antigenic load due to tobacco smoke or have an increased colonisation of the airways with microorganisms that could stimulate an IgE response [Burrows B, Halonen M, Barbee RA, Lebowitz MD. (1981). The relationship of serum immunoglobulin E to cigarette smoking. The American Review of Respiratory Disease. 124:523-5]. Haematologic malignancy interesting Th2 limphocytes line, such as Hodgking’s disease, can induce, through IL-4 secretion, IgE production [Zagami F., Esposito C., Tarro G., German A.M. (1992) IgE – Ferritina: Indicatori di recidiva nel morbo di Hodgkin. Agg. Med.&Chir. 10, 1, 76-88 – [Only registered and activated users can see links. Click Here To Register...]. Same condition is showed in non-sensitised pregnant where a more pronounced Th2 deviation is verified during pregnancy with elevated production of Th2-like cytokines and a decrease in Th1-like cytokines during normal pregnancies and subsequent total IgE increase. More, increased total-IgE in cord blood (cIgE) cannot be used as an indicator to single outhigh risk infants from atopy family history [Sybilski A.J., Doboszynska A., Samolinski B. (2009) Prediction of atopy in the first year of life using cord blood IgE levels and family history. Eur. J. Med. Res. 14(Suppl. IV): 227-232]. At this aspecificity is associated a reported inadequate sensitivity [Carosso A., Bugiani M., Migliore E., Ant J.M., De Marco R. (2007) Reference values of total serum IgE and their significance in the diagnosis of allergy in young European adults. Int Arch Allergy Immunol 142: 230-238]. So, total IgE cannot be used to rule out the presence of sensitization to common inhalant allergens in adult subjects with symptoms suggesting allergy. Its role as part of a routine diagnostic work up in patients suspected of having allergic disease is therefore limited [M. Kerkhof M., Dubois A. E. J., Postma D.S., Schouten J.P., de Monchy J. G. R. (2003) Role and interpretation of total serum IgE measurements in the diagnosis of allergic airway disease in adults. Allergy 58: 905–911]. In conclusion total serum IgE is quite variable in allergic patients, also with normal levels, and decision making for doing further investigation should not be based on the result of total IgE, so not to recommend its measurement as the routine evaluation.

Zagami 01-07-2014 07:36 PM

Re: Allergen testing
 
Useful of total IgE determination in allergic diseases
Measurement of total IgE, in serum, secretion or on cell surfaces is of little diagnostic value. The reason is that mitogenic factors in viruses (e.g., Cytomegalovirus - CMV), bacteria (e.g., Staphylococcus), helminths (e.g., Ascaris, Schistosoma) and adjuvant factors in air pollution (e.g., cigarette smoke, and diesel exhaust) stimulate the production of IgE molecules without initiating any allergen specific IgE-sensitization. For the last issue it has been theorised that smokers may have a higher than average antigenic load due to tobacco smoke or have an increased colonisation of the airways with microorganisms that could stimulate an IgE response [Burrows B, Halonen M, Barbee RA, Lebowitz MD. (1981). The relationship of serum immunoglobulin E to cigarette smoking. The American Review of Respiratory Disease. 124:523-5]. Haematologic malignancy interesting Th2 limphocytes line, such as Hodgkings disease, can induce, through IL-4 secretion, IgE production [Zagami F., Esposito C., Tarro G., German A.M. (1992) IgE Ferritina: Indicatori di recidiva nel morbo di Hodgkin. Agg. Med.&Chir. 10, 1, 76-88 [Only registered and activated users can see links. Click Here To Register...]. Same condition is showed in non-sensitised pregnant where a more pronounced Th2 deviation is verified during pregnancy with elevated production of Th2-like cytokines and a decrease in Th1-like cytokines during normal pregnancies and subsequent total IgE increase. More, increased total-IgE in cord blood (cIgE) cannot be used as an indicator to single outhigh risk infants from atopy family history [Sybilski A.J., Doboszynska A., Samolinski B. (2009) Prediction of atopy in the first year of life using cord blood IgE levels and family history. Eur. J. Med. Res. 14(Suppl. IV): 227-232]. At this aspecificity is associated a reported inadequate sensitivity [Carosso A., Bugiani M., Migliore E., Ant J.M., De Marco R. (2007) Reference values of total serum IgE and their significance in the diagnosis of allergy in young European adults. Int Arch Allergy Immunol 142: 230-238]. So, total IgE cannot be used to rule out the presence of sensitization to common inhalant allergens in adult subjects with symptoms suggesting allergy. Its role as part of a routine diagnostic work up in patients suspected of having allergic disease is therefore limited [M. Kerkhof M., Dubois A. E. J., Postma D.S., Schouten J.P., de Monchy J. G. R. (2003) Role and interpretation of total serum IgE measurements in the diagnosis of allergic airway disease in adults. Allergy 58: 905911]. In conclusion total serum IgE is quite variable in allergic patients, also with normal levels, and decision making for doing further investigation should not be based on the result of total IgE, so not to recommend its measurement as the routine evaluation.


All times are GMT. The time now is 07:27 AM.

Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved

Page generated in 0.10090 seconds with 11 queries