Tag Archives: ELISA

Gabriela Lopez, 3M Food Safety
Allergen Alley

Method Acting: Comparing Different Analytical Methods for Allergen Testing and Verification

By Gabriela Lopez-Velasco, Ph.D.
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Gabriela Lopez, 3M Food Safety

Every day, food industries around the world work to comply with the food labeling directives and regulations in place to inform consumers about specific ingredients added to finished products. Of course, special attention has been placed on ensuring that product packaging clearly declares the presence of food allergens including milk, eggs, fish, crustacean shellfish, tree nuts, peanuts, wheat, soy, sesame and mustard. (Additional food allergens may also be included in other regions.)

But labeling only covers the ingredients deliberately added to foods and beverages. In reality, food manufacturers have two jobs when it comes to serving the needs of their allergic consumers:

  1. Fully understand and clearly declare the intentional presence of allergenic foods
  2. Prevent the unintended presence of allergenic foods into their product

Almost half of food recalls are the result of undeclared allergens, and often these at-fault allergens were not only undeclared but unintended. Given such, the unintended presence of allergenic foods is something that must be carefully considered when establishing an allergen control plan for a food processing facility.

How? It starts with a risk assessment process that evaluates the likelihood of unintentionally present allergens that could originate from raw materials, cross-contact contamination in equipment or tools, transport and more. Once the risks are identified, risk management strategies should then be established to control allergens in the processing plant environment.
It is necessary to validate these risk management strategies or procedures in order to demonstrate their effectiveness. After validation, those strategies or procedures should then be periodically verified to show that the allergen control plan in place is continually effective.

In several of these verification procedures it may be necessary to utilize an analytical test to determine the presence or absence of an allergenic food or to quantify its level, if present. Indeed, selecting an appropriate method to assess the presence or the level of an allergenic food is vitally important, as the information provided by the selected method will inform crucial decisions about the safety of an ingredient, equipment or product that is to be released for commercialization.

A cursory review of available methods can be daunting. There are several emerging methods and technologies for this application, including mass spectroscopy, surface plasmon resonance, biosensors and polymerase chain reaction (PCR). Each of these methods have made advancements, and some of them are already commercialized for food testing applications. However, for practical means, we will discuss those methods that are most commonly used in the food industry.

In general, there are two types of analytical methods used to determine the presence of allergenic foods: Specific and non-specific methods.

Specific tests

Specific methods can detect target proteins in foods that contain the allergenic portion of the food sample. These include immunoassays, in which specific antibodies can recognize and bind to target proteins. The format of these assays can be quantitative, such as an enzyme-linked immunosorbent assay (ELISA) that may help determine the concentration of target proteins in a food sample. Or they can be qualitative, such as a lateral flow device, which within a few minutes and with minimum sample preparation can display whether a target protein is or is not present. (Note: Some commercial formats of ELISA are also designed to obtain a qualitative result.)

To date, ELISA assays have become a method of choice for detection and quantification of proteins from food allergens by regulatory entities and inspection agencies. For the food industry, ELISA can also be used to test raw ingredients and final food products. In addition, ELISA is a valuable analytical tool to determine the concentration of proteins from allergenic foods during a cleaning validation process, as some commercial assay suppliers offer methods to determine the concentration of target proteins from swabs utilized to collect environmental samples, clean-in-place (CIP) final rinse water or purge materials utilized during dry cleaning.

ELISA methods often require the use of laboratory equipment and technical skills to be implemented. Rapid-specific methods such as immunoassays with a lateral flow format also allow detection of target specific proteins. Given their minimal sample preparation and short time-to-result, they are valuable tools for cleaning validation and routine cleaning verification, with the advantage of having a similar sensitivity to the lowest limit of quantification of an ELISA assay.

The use of a specific rapid immunoassay provides a presence/absence result that determines whether equipment, surfaces or utensils have been cleaned to a point where proteins from allergenic foods are indiscernible at a certain limit of detection. Thus, equipment can be used to process a product that should not contain a food allergen. Some commercial rapid immunoassays offer protocols to use this type of test in raw materials and final product. This allows food producers to analyze foods and ingredients for the absence of a food allergen with minimum laboratory infrastructure and enables in-house testing of this type of sample. This feature may be a useful rapid verification tool to analyze final product that has been processed shortly after the first production run following an equipment cleaning.

Non-Specific Tests

While non-specific testing isn’t typically the best option for a cleaning validation study, these tests may be used for routine cleaning verification. Examples of non-specific tests include total protein or ATP tests.

Tests that determine total protein are often based on a colorimetric reaction. For example, commercial products utilize a swab format that, after being used to survey a defined area, is placed in a solution that will result in a color change if protein is detected. The rationale is that if protein is not detected, it may be assumed that proteins from allergenic foods were removed during cleaning. However, when total protein is utilized for routine verification, it is important to consider that the sensitivity of protein swabs may differ from the sensitivity of specific immunoassays. Consequently, highly sensitive protein swabs should be selected when feasible.

ATP swab tests are also commonly utilized by the food industry as a non-specific tool for hygiene monitoring and cleaning verification. However, the correlation between ATP and protein is not always consistent. Because the ATP present in living somatic cells varies with the food type, ATP should not be considered as a direct marker to assess the removal of allergenic food residues after cleaning. Instead, an analytical test designed for the detection of proteins should be used alongside ATP swabs to assess hygiene and to assess removal of allergenic foods.

Factors for Using One Test Versus Another

For routine testing, the choice of using a specific or a non-specific analytical method will depend on various factors including the type of product, the number of allergenic ingredients utilized for one production line, whether a quantitative result is required for a particular sample or final product, and, possibly, the budget that is available for testing. In any case, it is important that when performing a cleaning validation study, the method used for routine testing also be included to demonstrate that it will effectively reflect the presence of an allergenic food residue.

Specific rapid methods for verification are preferable because they enable direct monitoring of the undesirable presence of allergenic foods. For example, they can be utilized in conjunction with a non-specific protein swab and, based on the sampling plan, specific tests can then be used periodically (weekly) for sites identified as high-risk because they may be harder to clean than other surfaces. In addition, non-specific protein swabs can be used after every production changeover for all sites previously defined in a sampling plan. These and any other scenarios should be discussed while developing an allergen control plan, and the advantages and risks of selecting any method(s) should be evaluated.

As with all analytical methods, commercial suppliers will perform validation of the methods they offer to ensure the method is suitable for testing a particular analyte. However, given the great diversity of food products, different sanitizers and chemicals used in the food industry, and the various processes to which a food is subjected during manufacturing, it is unlikely that commercial methods have been exhaustively tested. Thus, it is always important to ensure that the method is fit-for-purpose and to verify that it will recover or detect the allergen residues of interest at a defined level.

Eurofins Technologies, Gold Standard Diagnostics

Eurofins Technologies, Gold Standard Diagnostics Enter Strategic Partnership

Eurofins Technologies, Gold Standard Diagnostics

This week Eurofins Technologies announced a strategic partnership with Gold Standard Diagnostics (GSD), a developer and manufacturer of fully-automated diagnostic instruments and assays for various test methods. The agreement unites Gold Standard’s ELISA-based instruments and Eurofins Technologies rapidly-expanding diagnostic test kit portfolio for  food, environmental and animal health testing.

Gold Standard Diagnostics will be the standard platform for Eurofins Technologies ELISA-based food testing kits including food pathogens, allergens, mycotoxins, veterinary drug residues, and animal health kits.

Dollar

Pathogens Drive More Than Half of $12 Billion Global Food Safety Testing Market

By Maria Fontanazza
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Dollar

The importance of food safety testing technologies continues to grow, as companies are increasingly testing their products for GMOs and pesticides, and pathogens and contamination. Last year the global food safety testing market had an estimated value of $12 billion, according to a recent report by Esticast Research & Consulting. Driven by pathogen testing technologies, the global food safety testing market is expected to experience a 7.4% CAGR from 2017–2024, hitting $21.4 billion in revenue in 2024, said Vishal Rawat, research analyst with Esticast.

With a CAGR of 9.3% from 2017–2024, rapid testing technologies are anticipated to lead the market. Testing methods responsible for this growth include immunoassays (ELISA), latex agglutination, impedance microbiology, immune-magnetic separation, and luminescence and gene probes linked to the polymerase chain reaction, said Rawat, who shared further insights about the firm’s market projections with Food Safety Tech.

Food Safety Tech: With the GMO food product testing market expected to experience the highest growth in the upcoming future, can you estimate the projected growth?

Vishal Rawat: The GMO food product testing market is estimated to generate a revenue of approximately $5.2 billion in 2016. The market segment is expected to witness a compound annual growth rate of 8.3% during the forecast period of 2017–2024. This is a global market estimation.

FST: What innovations are occurring in product testing?

Rawat: Nanomaterials and nanobased technologies are attracting interest for rapid pathogen testing. Sustainable technologies such as edible coatings or edible pathogen detection composition can attain a trend in the near future. Also, new rapid allergen testing kits are now emerging out as the latest food testing technology in the market, which are portable and easy to use.

FST: Which rapid pathogen detection testing technologies will experience the most growth from 2017–2024?

Rawat: New and emerging optical, nano-technological, spectroscopic and electrochemical technologies for pathogen detection, including label-free and high-throughput methods would experience the highest growth.

FST: What pathogen testing technologies are leading the way for meat and poultry in the United States?

Rawat: The presence of a microbial hazard, such as pathogenic bacteria or a microbial toxin, in ready-to-eat (RTE) meat or poultry products is one basis on which these products may be found adulterated. The FSIS is especially concerned with the presence of Listeria monocytogenes, Salmonella, Escherichia coli O157: H7, and staphylococcal enterotoxins in RTE meat and poultry products. Rapid pathogen testing for E. coli O157:H7 and Salmonella, for ground beef, steak and pork sausages is going to lead the U.S. market.

An overview of the report, “Food Safety Testing Market By Contaminant Tested (Pathogens, GMOs, Pesticides, Toxins), By Technology (Conventional, Rapid), Industry Trends, Estimation & Forecast, 2015– 2024” is available on Esticast’s website.

Suresh Neethirajan, University of Guelph
In the Food Lab

Identifying Peanut and Other Allergens Outside the Lab

By Suresh Neethirajan, Ph.D
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Suresh Neethirajan, University of Guelph

Judging the nature and suitability of items we put in our mouths is a task we perform daily, whether it’s due to different taste preferences, being on a diet, or from particular foods not agreeing with our metabolisms. Some foods can trigger mild reactions such as an upset stomach, or more serious skin rashes and outbreaks, from shortness of breath to even death.

Many of us have been somewhere where someone with a peanut allergy has been brought to everyone’s attention. The situation may have been publicized before boarding a plane, at a school where parents are asked to refrain from giving their children any food containing peanut products, or restaurants that clearly indicate which dishes are peanut-free on their menu, or that the kitchen is absent of the legume.

The number of people with food allergies continues to rise, and although many theories have been provided for the increase, the exact cause is unknown. Many foods are documented as being able to produce an allergic reaction—milk, eggs, soy and shellfish, to name a few—but peanuts and gluten are highlighted as major offenders. Canadian government regulations require that manufacturers label products that contain certain allergens, even if they are made in a facility where allergens are in another product.

The Threat of Gluten and Peanuts

Gluten contained in wheat has become a widely avoided food substance, although the reason for this might has more to do with health concerns than allergies. The American College of Allergy, Asthma and Immunology (ACAAI) estimates that 400,000 U.S. school children have a peanut allergy, with many of those also having other food allergies. According to the ACAAI, many children will eventually outgrow most food allergies, but only 20% of those who have a peanut tolerance will outgrow it.

The charity organization Food Allergy Canada states that 2.5 million people suffer from a food allergy in Canada, while 2 in 100 children are susceptible to peanuts causing a reaction. There isn’t a cure for food allergies, so governments and food inspectors have the weighty task of ensuring that commercially produced products are packaged or served with proper labeling and information to protect consumers. This requires constant checking and testing of products that may have come in contact with peanuts or gluten.

New Tool for Food Inspectors

To provide regular analysis, the procedure has been lengthy and expensive, but scientific researchers at Canada’s University of Guelph have developed an apparatus that can identify allergens in a much shorter time span while being considerably more cost effective. The new allergen detector could expedite allergen reporting and possibly reduce the number of allergic reactions through more timely results.

Biosensor, University of Guelph
Schematic of the biosensor for the rapid detection of food allergens. Image courtesy of BioNanoLab, University of Guelph.

Based on the ELISA (enzyme-linked immunosorbent assay) platform that is widely used in diagnostic labs to identify allergens, the new apparatus provides comparable accuracy. The technology has been miniaturized so that equipment is portable, about the size of an audiocassette case, and tests can be conducted on location instead of relying on a lab that may be far away.

An Allergen that Glows

In the case of peanuts, the scientists focused on a prominent allergen named Ara h 1, because it can be identified through non-radioactive fluorescence. Although there are other allergens in peanuts, they don’t share the same property by which they can be identified, as does Ara h 1.

The process requires a small amount of the suspected food to be liquefied in a suspension so that it can be injected using a filter syringe into a silicon-based plate, or chip, of microcapillaries. As the sample passes through tiny tubes of the microfluidic chip using capillary action, it travels through a beam of light from a LED source that is monitored by a specialized camera, which is also a product of the scientists’ work.

The image captures Ara h 1 protein particles that fluoresce when they come in contact with the chemical properties of the suspension. Currently, the camera records the data and sends it to a computer to be analyzed and deciphered with a result being provided within 20 minutes, compared to a conventional lab test that takes up to four hours after a sample has been received.

In a modification to provide an extremely portable system, research is underway to develop an app to enable results via a smartphone. Testing foods in the near future will be as convenient and prompt as holding the detector in one hand and a smartphone in the other so that a restaurant owner, for example, will be assured that dishes are allergen-free before being served to customers.

Imitating the Human System for Detection

To enable the allergen to fluoresce, the compound graphene oxide (GO) was utilized in combination with a bio-sensing component, known as an aptamer. The aptamer acts similarly to antibodies that identify and attach themselves to foreign and hostile elements that enter our blood system. Once a GO-aptamer mixture is attached to the allergen, the light source allows the protein particle to be detected and its image captured electronically.

By altering an aptamer’s composition to identify other allergens, such as gluten, the detector is a versatile piece of scientific equipment for identifying potentially hazardous food ingredients. The developers of the technology are confident that their discovery will change the future of identifying potentially hazardous food components. The final step in the allergen detector’s development seems to be fine tuning the detection process for certain processed foods, such as roasted peanuts, that can alter the composition of Ara H 1 making it less obvious to be identified.