ELISA Tests: The Basics
Aquanette currently serves as Sr. Manager, R&D at Nima Labs. She has more than 15 years of experience in assay development, validation and optimization. Her experience includes molecular biology, in vitro diagnostics, protein-based diagnostics, reagent formulation, and immunoassay development including ELISA. Her experience has spanned basic research to FDA regulated product development and maintenance/troubleshooting.
When researching food testing and allergen detection, you may have come across the acronym ELISA. But what is an ELISA test exactly? Considering ELISA tests are commonly used in food testing, we compiled some information to help shed light on how they work and how they fit into the food testing industry landscape.
What does ELISA stand for?
Enzyme-Linked ImmunoSorbent Assay.
What is an ELISA test?
An ELISA is a type of assay (or test) used to detect different types of biological molecules, such as peptides, proteins, and antibodies.
ELISA tests are good tools for food allergen detection because they give you the ability to isolate desired materials, amplify detection using enzymes, and quantify how much of a particular allergen is present.
If your goal is to be able to identify the amount of an allergen in a sample of food, an ELISA test is one way to do this.
Who uses ELISA tests?
Food manufacturers that need to test raw materials or finished goods, often use ELISA. They typically have a testing team in house or contract with a certified lab to do the testing for them.
The reason why ELISA tests are mainly used by food manufacturers, is because it requires special lab equipment and specific training that is not typically available to the general public.
Who makes ELISA tests?
For a given allergen, such as gluten or peanut, there are many different ELISA test kits in use by different labs, produced by many different companies. Kit components such as: antibodies, extraction buffer, wash buffer & recommended incubation times, can all differ across different manufacturer’s kits.
How do ELISA tests work?
In the allergen testing industry the “sandwich ELISA” is most common. In a sandwich ELISA, the allergen of interest is sandwiched between two antibodies – the antibodies make the bread, the allergen the filling. There is a capture antibody and a conjugate antibody.
There are many variants to this scenario but generally the steps are as follows:
- Prepare materials for the test
- Extracted food sample in buffer solution
- Kit components – well plate coated with capture antibody, wash buffer, conjugate, substrate, and stop solution
- Add extracted food sample solution to well plate
- Add conjugate antibody to well plate
- Add substrate
- If the enzyme substrate “lights up” the allergen is present at some level
- Stop enzyme/substrate reaction to prevent over development
- Read well plate (i.e. measure signal using specialized equipment)
- Data analysis and interpretation
Want to know more? Here’s a handy youtube video showing the whole process.
Are there any issues with ELISA testing?
Currently, the FDA does not regulate any ELISA used for allergen testing in food, as they are not considered medical devices. Similarly the FDA does not recommend a particular kit over another. As with any analytical test, there can be differences between kits. Two of the main causes of variation are:
1. Antibodies used:
Kit manufacturers usually have proprietary antibodies that may perform differently in their ability to “capture” allergens. As food can be prepared in many ways (raw, boiled, fried) the allergen contained therein can “look” different and may not be as efficiently captured.
2. Extraction buffer used:
The extraction buffer is used to get the allergen of interest out of the food sample and into the solution used for the ELISA test. Kit manufacturers usually have proprietary buffers that work within their assays, but not necessarily in other assays. In addition to manufacturer differences, these extraction buffers differ in the ability to get the same amount of allergen from food samples with varying ingredients, textures, and consistencies. For example, oatmeal that has had 20 ppm of gluten added might give a different ELISA reading than a muffin spiked to the same 20 ppm level. Furthermore, a pasta tested with one kit can return a value of 13.2 and a value of 27.6 on a different kit (Evaluation of a Handheld Gluten Detection Device).
3. Lack of a universal standard across kits
A further limitation is a lack of a universal standard across kits – there is no one calibrator, or set of calibrators, used collectively by kit manufacturers. Each kit manufacturer calibrates, or quantifies, with reference materials that may vary in buffer composition, as well as the protein used to determine concentration.
To address some of the variance seen from kit to kit, labs generally participate in proficiency sample testing. Proficiency sample testing assesses both a lab and the kit’s ability to return a ppm value for a given sample that is within range of reported values for the sample. In other words, they are used as a measure of whether the kit (and lab) is functioning properly. Results can vary by as much as 50%.
Although ELISA is considered quantitative, these varying proficiency test results make it more difficult to standardize ELISA results across kits and food matrix.
As with all immunoassays, ELISA can be limited in its use due to: only sampling of a portion of food, unintended interference or cross-reactivity to foods or ingredients, pH, food preparation and processing.
Is Nima an ELISA test?
The short answer: no. Nima is often compared to ELISA, so let’s look at some of the similarities and differences.
ELISA and Nima are both antibody based and use “detection molecules” to determine if there is allergen in a sample.
As you saw above, ELISA is a complex process with many steps, that requires special training and “costly” equipment that is not available to most consumers. Consumers can, however, send out samples to labs equipped for testing with ELISA. The upside: the testing takes place in a controlled lab environment. The downside: this takes time and money, so it may not be helpful for a consumer whose goal is to know whether the dish in front of them is safe.
Nima testing does not take place inside a lab. But it also does not require special equipment or training. It only entails a 4 step process (at most) from the user, because the sensor and capsule do the majority of the testing process without needing the user to take any action. Because of this you are able to test and get results quickly, in real world contexts (like a potluck dinner at your friend’s house).
Different intended purpose
The intended purposes of ELISA and Nima are very different.
ELISA is used by professionals, in a controlled lab setting with the intent of quantifying a sample. It’s a great tool for evaluating levels of allergens in food – which is why it is often used by food manufacturers to determine whether or not they are in compliance with regulatory standards.
Nima is used by anyone, in the wild. The purpose is not to measure whether a sample of food has 5 ppm of an allergen versus 10 ppm. The purpose is to give someone the chance to catch unwanted allergens in their food, before they eat. It is meant to be an extra piece of information to help people manage their risks, in a real world environment.
ELISA tests are widely used throughout the food testing industry. While robust and quantitative, ELISA testing is complicated and requires a high level of experience to run and interpret correctly. There is a huge value in these tests being available to labs and they serve an important purpose in the food production industry. However there are limitations in the way that ELISA technology can be applied to the everyday lives of the average person. We look forward to seeing continued development of technologies and testing methods to help keep the free-from community safe as they navigate their everyday lives.