Peanut allergen quantification: a tough nut to crack

As part of the National Measurement Laboratory’s 30th anniversary, we’re sharing stories and case studies from the last three decades.

One of our case studies touches on an issue that affects hundreds of thousands of people across the UK alone: peanut allergies. So read on to learn how LGC scientists developed a unique allergen quality control material, which can be used to help protect the people in the UK with a peanut allergy and also help to prevent contamination in the food production process, potentially saving the food industry millions of pounds.

The problem

The prevalence of peanut allergy has nearly doubled in Europe over the past two decades and it now affects around 500,000 people in the UK [1]. Peanut allergy is the most common cause of fatal food allergy reaction. It occurs when the immune system mistakenly identifies peanut proteins as something harmful. The fear of accidental exposure in food reduces the quality of life of peanut allergy sufferers and severely limits the social habits of allergic individuals, their families and even their friends.

SMALL_iStock_000004018390Small peanutsIt is not only those with peanut allergies who have to worry about the risk of allergic reactions or death by anaphylaxis; it also creates problems for businesses. Testing for allergen proteins in food is difficult, as samples usually contain a lot of protein and it can be difficult to separate the allergen protein of interest. This has an impact on the ability of manufacturers and suppliers to adequately label their goods and also has implications for defining threshold levels and detecting food fraud.

All food companies throughout the EU are compelled by law to declare major allergens including peanut, if included in food products as ingredients. The current labelling rules, brought into force in December 2014 by European Regulation 1169/2011 (the EU Food Information for Consumers Regulation, EU FIC) ensure that all consumers are given highlighted information about the use of allergenic ingredients in pre-packed food [2]. This is to make it easier for people with food allergies to identify the foods they need to avoid. The EU FIC also extends to food sold loose or served when eating out. Prevention of cross contamination with peanut through product testing, validation and verification of cleaning, and checking of ‘peanut-free’ products requires exacting testing.

ELISA (enzyme-linked immunosorbent assay), PCR (polymerase chain reaction) and mass spectrometry (MS) methods can be used to detect food allergens, but there are problems obtaining reliable quantitative results with all three. Prior to this project, there were no suitable reference materials available in the form of a food matrix, making it difficult for laboratories and test-kit manufacturers to validate quantitative methods for allergen measurement.

The solution

A quality control (QC) material that is a real food, containing a known amount of specific allergen protein, and is stable and homogenous could assist laboratories in the validation and monitoring of their analysis. Consequently, a project was undertaken by LGC to develop a food matrix peanut allergen QC material.

The chosen matrix was a chocolate dessert product developed for low-dose threshold studies in food allergic individuals in the European research project ‘EuroPrevall’. Two QC materials were prepared by University of Manchester researchers in the form of chocolate dessert product pastes designed to be reconstituted with water before analysis. One material (LGCQC1011) was prepared as a peanut free negative control and the other material (LGCQC1012) was prepared as a positive control with the addition of light roast, partially defatted peanut flour (a commercial food ingredient) to give a peanut protein content of 10 mg kg-1. The pastes were transferred to LGC, packaged in nitrogen-flushed sealed sachets to aid stability and the units were numbered sequentially in fill order. LGC assessed and proved their homogeneity and stability, underpinned by a validation study of the test method using a commercially available ELISA kit (Romer AgraQuant® Peanut kit). The National Measurement System funded the ELISA kit validation studies, and a Technology Strategy Board and LGC co-funded research and development project established the design and production of the QC material.

Impact

Failure in food allergen management means ‘food-allergen’ related incidents are the most common reason for product withdrawals and recalls in the United Kingdom according to the UK Food Standards Agency. The 34 recalls related to allergens in 2010 were estimated to cost stakeholders £10- 15 million. In 2013, the number of Allergy Alerts issued to withdraw food or drink products had risen to 47.

Phil Goodwin, MD of Bio-Check (UK) a food allergen test kit manufacturer, has worked in this area for 30 years and welcomes LGC’s recent initiatives:

“The science of food allergen detection, let alone quantitation, has failed to move forward anything like quickly enough since it began in the late 1980s. The emergence of such high quality QC materials as are being produced by LGC is a significant step forward to a time when all commercial test kits can be demonstrated to show good agreement on allergen levels. LGC are to be applauded for taking on this difficult challenge and I urge all allergen kit producers and analysts to use the material to improve their products and results.”

 

[1] http://www.mrc.ac.uk/news-events/publications/outputs-outcomesand-impact-of-mrc-research-2013-14/

[2] http://allergytraining.food.gov.uk/english/rules-and-legislation/

This blog first appeared as a NML case study on the LGC Group website. To learn more about the NML, visit their site here.

What’s funny about your honey?

Ensuring the safety and authenticity of the food we eat is of paramount importance and there is growing concern, both at the EU and global level, to ensure the quality control of food to protect the health and safety of consumers. And during the National Measurement Laboratory’s thirty years, we’ve done a lot of work to support reliable measurements in food testing and authentication.

Honey is known to have multiple health and nutritional benefits and is in high demand among consumers. It is defined as the natural sweet substance produced by bees and there is significant regulation around the composition and labelling of honey in order to protect consumers from food fraud. However, due to the declining numbers of bees, the impact of weather conditions on supply and the high costs production, honey is expensive. This makes it a prime target for economically-motivated food fraud.

StockSnap_97LJAKWL36Some research suggests that humans began to hunt for honey 8,000 years ago, and the oldest known honey remains, dating back to between 4,700 – 5,500 years ago, were discovered in clay vessels inside of a tomb in the country of Georgia.

The ancient Egyptians used honey to sweeten dishes and to embalm the dead, while the ancient Greeks actually practised beekeeping so much that laws were passed about it. Honey was prevalent around the ancient world, being used in ancient India, China, Rome and even among the Mayans. It even plays a role in many religions, representing the food of Zeus, an elixir of immortality, and a healing substance.

And just like any other important product, fraudsters have been faking it since it’s been in use. Ancient Greeks and Romans both mention honey adulteration, and back in 1889, Dr Harvey W. Wiley testified in front of Congress that it was the most adulterated product in the U.S.

Honey is still one of the most adulterated food products globally, with a report last year citing that more than 14% of tested samples were adulterated.

There are two types of food fraud associated with honey: adulteration and fraudulent labelling. Honey adulteration typically occurs by substituting honey for cheaper sweeteners such as high fructose corn syrup, cane or beet sugar syrup. Fraudulent labelling occurs because honeys from a particular geographic or botanical source, such as Manuka, command premium prices amongst consumers.

Detecting these types of fraud presents a significant measurement challenge for food regulators: adulterated products show very similar physical and chemical properties to pure honey and mis-labelled products are, in fact, pure honey, just of lower quality. Several reports indicate that there is more Manuka honey being sold than Manuka bees can  produce, which illustrates how often lower quality honeys are passed for premium ones in order to maximise profit.

During our thirty years as the National Measurement Laboratory (NML) for chemical and bio-measurement, our scientists have conducted several reviews and studies of methods for detecting honey fraud1. For instance, nearly forty years ago, scientists began to use stable carbon isotope ratio mass spectrometry (IR-MS) to detect high fructose corn syrup in honey.  As our scientists found2, it is possible to identify food fraud in honey using IR-MS, which measures small but observable variations in the ratios of the two stable isotopes of carbon (C-13 and C-12). Sugars, although chemically identical, have a different isotopic signature depending on the way in which the plant processes carbon dioxide. As the majority of honey-source plants use a different pathway than plant sugars typically used as honey adulterants, it is possible to detect adulteration using IR-MS. The specific geography of the plants also plays a role in the isotopic fingerprint and IR-MS can be used to determine where honeys originated.

However, in order that these types of measurements are robust and reliable in detecting food fraud across the supply chain the comparability of results is critical. To support this, LGC co-ordinated an international comparison study in 2016 for isotope ratios in honey involving 6 national measurement institutes (NMIs) and 6 expert laboratories (contacted via the Forensic Isotope Ratio Mass Spectrometry (FIRMS) Network) and the results between participants showed good comparability.

Demonstrating the comparability of isotope ratio measurements is crucial to detecting many types of food fraud and supporting food authenticity claims, of which honey is just one example. The international study coordinated by LGC demonstrates the measurement framework is in place to support food fraud regulation in the future.

 

1 D. Thorburn Burns, Anne Dillon, John Warren, and Michael J. Walker, 2018, A Critical Review of the Factors Available for the Identification and Determination of Mānuka Honey, Food Analytical Methods, https://doi.org/10.1007/s12161-018-1154-9.

2 Helena Hernandez, “Detection of adulteration of honey: Application of continuous-flow IRMS”, VAM Bulletin, 1999, Vol 18, pp 12-14.

The National Measurement Laboratory turns 30!

In 1988, Government Chemist Alex Williams, seeing the need for improved quality of analytical measurements, initiated and launched the Valid Analytical Measurement (VAM) programme to develop a chemical measurement infrastructure in the UK.

This programme would go on to evolve into the National Measurement Laboratory for chemical and bio-measurement. The UK was one of the pioneers within the global measurement community to recognise the need to address the new and developing challenges of measurement across chemistry and biology.

An article from the early VAM bulletins (1989).

That means 2018 marks the NML’s 30th birthday and kicks off our ‘Year of Measurement’. It is an opportunity to celebrate the importance of measurement science (‘metrology’) as we enjoy our 30th birthday and join the upcoming Festival of Measurement, which launches in September and lasts through May 2019.

In our thirty year history of performing measurements to support the UK, we’ve experienced a lot of growth, seen big changes in the challenges we’ve been set and made some major breakthroughs. We’ve asked (and answered) a lot of questions, like ‘What are the best methods for the detecting the adulteration of honey’ or ‘Is the computer a friend or foe?’ (The answer is ‘friend’…or ‘both’ if you’ve invested heavily in encyclopaedias.)

We’ve already outlined in a recent blog post how important accurate measurement is, affecting everything from food and drink to medicine. Accurate and precise measurement is the foundation of public health and safety. But it’s also just as important to the economy.  In 2009, it was estimated that £622 billion of the UK’s total trade relied on measurement in some way, meaning that measurement plays a role in nearly every aspect of our lives.

Our Chief Scientific Officer, Derek Craston, agrees that good measurement is crucial to economies. ““In my role, I am fortunate to be able to see the major benefits that chemical and biological measurements make to the prosperity of companies and the lives of individuals across areas as broad as clinical diagnosis, drug development, environmental protection and food security. Indeed, in a global economy, with complex supply chains and regulatory frameworks, it is hard to see how many markets could function without it.”

We’re proud of the work we’ve done as the National Measurement Laboratory, where our work supports manufacture and trade, protects consumers and enhances quality of life. And over the next few months, we plan to share stories and case studies from our thirty years at the forefront of measurement with you, as well as look forward to the next thirty years.

Delivering impact to support AIDS research

LGC is helping to ensure that research into a cure for HIV is based on sound fundamental measurements.

Over 36 million people currently live with HIV, with approximately 2 million becoming infected each year (WHO 2015). Although HIV can be successfully managed with combination antiretroviral therapy (cART), the therapy must be continued indefinitely as no cure presently exists. This can be challenging in regions with high HIV prevalence and long-term use can potentially have toxic side effects.

One barrier to curing HIV is the presence of infected host cells that are not targeted by current therapies but lay dormant (so-called ‘viral reservoir’). These cells have the potential to become re-activated so novel strategies to cure HIV aim to target this reservoir. To determine whether these new approaches are successful, accurate and robust, methods for measuring HIV DNA are required.

The Molecular and Cell Biology team at LGC perform research to support accurate and reliable measurement as part of our National Measurement Laboratory (NML) role. Recent work by NML scientists comparing different molecular methods (qPCR, digital PCR) for quantification of HIV DNA has raised some concerns around the current popular choice of calibrator used to compare results between HIV clinical studies (8E5, ATCC® CRL-8993). It appears to lose HIV DNA copies during cell growth, potentially producing misleading estimates of how much HIV DNA is present and affecting whether novel strategies towards curing HIV are deemed successful or not.

Based in part on our work, the NIH AIDS Reagent Program, which provides critical reagents and resources to support research in the areas of AIDS therapeutics and vaccine development, has recently highlighted the potential instability of the standard on its reagent database to support the research community and enable the best chances of success.

 

 

Citation:

Busby E et al. Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR (2017) Sci Rep 7(1):1209. doi:10.1038/s41598-017-01221-5

The importance of iodine – are you drinking enough milk?

Ensuring the safety of the food we eat is of paramount importance. Iodine is an essential element naturally found in some foods. Insufficient amounts of iodine in the diet results in low levels of thyroid hormones, which are responsible for regulation of metabolism.

In pregnant women and infants iodine is of particular importance as it plays a critical role in brain development. The primary sources of iodine for most people are milk and dairy products but due to increases in dairy intolerance and changes in diet, milk-products are being increasingly substituted for non-milk alternatives.

To identify the impact that such dietary changes might have on iodine levels across the population, an understanding of the levels of iodine naturally present in milk is necessary. This includes the effects of seasonal variations or fat content and any processing effects of pasteurisation which might reduce the iodine content. These variations have been investigated by the Nutrition Innovation Centre for Food and Health (NICHE), Ulster University, with milk samples collected over a 12-month period. However, these differences needed to be measured accurately in order to properly determine the influence different conditions have on iodine content.

As part of the UK’s National Measurement Laboratory (NML) role, scientists at LGC have developed a high accuracy quantitative method (inductively-coupled plasma mass spectrometry) for the analysis of iodine in milk and milk-products to support the regulation on iodine levels in infant formulas. Using this expertise, we were able to support the work being done at Ulster University, providing the analytical capability required to determine the levels of iodine in milk under a variety of conditions.

Of the collaboration, Maria O’Kane, lead author on the paper, said: “LGC facilitated my visit to the laboratory in Teddington and enabled me to undertake analysis of the milk samples collected using high accuracy ICP-MS. The expert staff at LGC supported my learning and enabled me to develop a greater knowledge and understanding of ICP-MS analysis.”

The findings were recently published in the Journal of Nutrition, where Maria concluded that consuming additional cow milk can significantly increase the amount of iodine observed in the urine of women of childbearing age.

This work will help our understanding of current iodine intake and support future research in this area and clearly demonstrates the impact the UK’s National Measurement Laboratory (NML) can have on real-world problems, protecting human health and ensuring the safety of our food.