Component Testing (Pt.2) - Why Should We Care?

So, in Part 1 of this labor of love, I talked about EIA vs. component testing for allergies. If you followed the science, you can start to see some situations where component testing might be helpful to figure out what's going on with our kids:

1. To identify cross-reactivity between food and pollen.

There are six peanut allergens that are currently the focus of component testing: Ara h 1, 2, 3, 5, 8 and 9. It's important to note that there are at least 24 identified, potentially allergenic epitopes just for Ara h 1!

The first three are what are known as seed storage proteins. They're important to the plant to ensure the next generation of little plants is grown. As a result, they're extremely durable and usually make it intact through break-down process in the stomach. Ara h 9 is a lipid transfer protein, responsible for moving fats across membranes within the cell. Again, it's a pretty hardy little beast.

In contrast, Ara h 5 and 8 are wimps. They also have homologues across many other plants, especially pollen from those plants, so the chances of cross-reactivity on a traditional allergen panel are high.

The traditional method of testing that uses a natural extract cannot tell these two types of results apart. One recent study showed that 11.8% of kids tested using a traditional RAST test showed up positive for peanut. However, when challenge tests were conducted, only a quarter of the kids actually reacted when they ate peanut. This roughly correlates with the estimated 1-2% rate of peanut allergy in the population.

The uKnow test is currently being marketed to parents whose kids had a positive peanut test but who have  never experienced an actual reaction to peanut. If you are in that category, this test could change your life.

Remember those look-alike proteins, or homologues? In the case of kids who test positive, but who really don't have a peanut allergy, there's a distinctive pattern that shows up:

Remember that Ara h 8 is a wimp. It mostly breaks down in the stomach to the point that the body doesn't react to it. That's why it often causes an itchy mouth but limited other symptoms.

This may be the explanation for the fabled "mild" peanut allergy. These kids will all have itchy mouths. Some of them may even have a few systemic symptoms. But, the thought is that they are not likely to tip over into serious anaphylaxis. (Of course, whether you're ready to turn in your epinephrine injector under those circumstances is between you and your doctor.)

Another great example of where the test is useful is my son's situation. His January RAST for hazelnut was ~3, yet the RAST just taken for the FAHF-2 study in April was a 12. That's a huge increase for a RAST in just four months!

It turns out that hazelnut (Cor a 1) and Birch (evil birch Bet v 1!) are strong homologues.¹ The large increase in his number during tree pollen season is a good indicator that we're probably looking at a less serious form of hazelnut allergy. We're hopeful that a component test can take this worry off his plate.

2. Possibly: to provide more information on the timeline to outgrow milk/egg allergy.

Milk and egg allergies are really tough on parents! For 17 years now, we've asked the question of our allergist that I'm sure all of you MA/EA parents ask each time: "when will my kid OUTGROW this allergy?!"

There are some things scientists have found out about these allergies. As with peanut, if kids are allergic to multiple allergenic proteins, it generally makes the allergy more persistent. Additionally, if a child's body can recognize epitopes when they're not folded up in a protein, that also means they're likely to have the allergy longer, maybe lifelong, and potentially have more serious reactions.

A traditional RAST test can crudely test for the different components of milk: whey, casein, α-lactalbumin, β-lactalbumin, but it can't identify specific epitopes and it can't demonstrate whether the allergy is to "folded" proteins (not as bad) or the "unfolded" strand of protein (bad). The component test, though, gets down to just the very small (sometimes just 10 amino acids) unfolded "hot spot" on the protein that's causing the problem.

For milk and egg, there are "hot spots" that have been associated with severe, persistent allergies. For egg, these hot spots are all on a protein called ovomucoid.²  For the persistent milk allergy kids, the epitopes were associated with the casein protein.³

So is this test really able to tell which kids will outgrow their milk and egg allergies? The answer is maybe. Some doctors are already using it to look at patterns in milk/egg and determine whether introducing baked milk will be helpful in speeding up the tolerance process. It's definitely something to ask your allergist about.

NOTE: Right now, ISAC (Immuno Solid Phase Allergen) testing is being done by one experimental laboratory (Phadia), with only 103 components from 47 allergens available.⁴  Your allergist may not have a relationship with this lab or be ready to roll this out to patients.

3. Future hope: to separate "potent" epitopes from less harmful ones and find the pattern for life-threatening allergies.

This is where we start to get into weighing the cost of the test against the information it provides. If you have a child who has already experienced a reaction to peanut, why might you want to have the test done?

So far, 95% of people who react to peanut in real life and who have had component testing show a sensitivity to Ara h 2. However, there also seems to be a correlation between how many epitopes of peanut you're allergic to and how allergic you are.⁵  The current theory for why is related to T cells, the first type of immune cell that responds to the allergen. (The Allergist Mom just did an awesome post on all the science behind this if you are interested.)

T cells are specific to the epitopes to which they bond, but the T cells all compete for the same pool of resources (the "antigen presenting cells" Allergist Mom mentions). Like fighting kids, they're fine if they think all the toys in the room are available to them, but when they see other kids who might want the same toys, they get upset and all hell breaks loose. So...the more epitopes a peanut-allergic person is sensitized to, the more likely it is that their reaction will be severe.⁶ 

However, so far there is no "smoking gun" epitope, or pattern of epitopes on any of the allergenic proteins, that signals potentially fatal reactions. Like fingerprints, the patterns from person to person can be very different. Scientists are still busily identifying and characterizing all the epitopes for peanut. Plus, different populations show different patterns of epitope sensitization, and even allergen sensitization (for example, Ara h 9 is more prevalent in Mediterranean populations). In order to answer these questions, they'll need a lot more data from people willing to take the test. However, those people taking the test will probably not benefit right away.

Are you willing to spend the money to get a picture of your kid's epitope "fingerprint", even though the data to understand what it means is not yet available? We're still thinking about this one.

4. Future hope: to help determine potential pitfalls for MFA kids.

When my son was around age 4, we went through a horrible period where we thought he had idiopathic anaphylaxis. Instead, it turned out that he had developed allergies to a wide range of legumes. The probable culprit? Ara h 1, which shares a seed pod protein with many other legumes.

The problem is that not all kids who are positive for Ara h 1 have allergies to other legumes! There's likely some other mechanism involved in sensitizing children to these look-alike proteins. (Remember the faces from the homologue example? Some people are just better at telling faces - and proteins - apart than others.)

This test has great potential to eventually tease out these linked proteins, far better than the crude botanical family relationships many of us use today. It's definitely not ready for prime time. But maybe someday soon, your doctor will be able to look at your child's peanut epitope pattern and make a good guess as to which tree nuts may or may not be a problem.

So...all in all, component testing is promising...but probably not 100% there yet for anything except for identifying those peanut allergies that are most likely cross-reactions to pollen. But what a gift this will be to those parents who are terrified their child is peanut allergic on the basis of testing only! And apparently, that's a good percentage of the kids out there.

PLEASE NOTE: these are MY thoughts on the value of this testing only. You should definitely discuss this with your physician. Additionally, I would love to hear from others interested in the science as to how they see this playing out in future.


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Component Testing (Pt. 1) - The Science

I've been seeing a lot of articles and comments lately about component testing. That's gotten me to thinking about under what circumstances it would be useful.

To explain what's new about component testing, I first have to explain what the current method of testing —  enzyme immunoassay — involves. (While I do have some background in the lab sciences field, I definitely welcome any corrections from actual scientists! Leave them as comments, please.)

EIA (sometimes called ELISA), is done using plates that are preloaded with the allergenic substance into a indentation called a well. Your doctor checks off the allergens she wants tested (egg, milk, peanut, etc.) and the technician puts a sample of blood from the test patient into the well that contains the appropriate allergen. The analyzer then sends the panel through a series of chemicals and processes that encourages the antibodies in the patient's blood to bond with the purified antigen/allergenic protein (like egg) that's in the well. The extra, unneeded gunk (technical term!) is washed away and then a chemical is added to make the bonded antibodies/antigens either change colors,  fluoresce (FIA) or become radioactive (RIA) so the analyzer can count them up.

The RAST (radioallergosorbent test) numbers we get back as a result is measures in kilounits per liter (kU/L), where the "unit" part is arbitrary. (One of the problems with early RAST testing is that the different analyzer manufacturers did not standardize on how they reported results, so result sets from two different analyzers could be very different.) This number is just telling us how much antibody from the patient's blood actually stuck to the plate.

The allergen extracts used in the current process are a little clunky. Laboratories extract the allergens used in the test panels from natural substances. This can be a big problem because, for example, using different varieties of apples or even a male vs. a female cat can result in variations in the amount of some proteins. The extracts are continually improved and modified, but different individuals or populations can be sensitive to different proteins, so the test is a one-size-fits-most process.

Additionally, proteins in the body and proteins in the lab don't always behave the same. For example, stomach acid can pull pieces of them apart, changing their three-dimensional shape and therefore how they bond. Other blood components (like plasma) can also alter how much antibody sticks to the plate. All of these things can contribute to false positives and false negatives on tests. That's why RAST test results have to be interpreted in conjunction with patient history and/or skin prick testing.

The "Ep" epitope areas on the protein hook up with
the "Ab" antibodies, triggering the immune response. 

Instead of whirling up a batch of peanut butter and purifying it to create an extract for a plate, component testing focuses very narrowly on just a small strand of protein called an epitope. These protein "hot spots" are the trouble makers on a food that cause the immune system to go nuts. A food protein may have just a few of these hot spots, or it may have dozens. And, to make things fun, a person may react to just one epitope within a protein or to several.

The enzyme immunoassay process used up until now contained ALL the appropriate epitopes because it started with a natural source. However, there was no way to tell which epitope or epitopes were causing the problem. In contrast, a component test uses a recombinant DNA process to make only a specific epitope. If the child is positive, he or she is positive only to that epitope.

Just like with faces, some bodies
are better at telling proteins apart 

There's another concept that can explain many of the false positives we experience with RAST testing: homologous proteins. Often just called homologues for short, these are look-alike proteins.

To give you an example, the birch protein (Bet v 1) looks like a whole lot of other proteins to the body, including those in celery, hazelnut and apple. Sometimes a primary allergy to birch pollen can cause a food allergy test to show a false positive. For many people, this cross-reaction only causes oral allergy syndrome (OAS) and their reaction never proceeds beyond an itchy mouth.

Sometimes homologues are harmless misunderstandings...and sometimes they mean a little bit more.  This study from 2004 shows that up to 40% of patients sensitized to birch pollen experienced more than just an itchy mouth when they ate peanut butter, even though their peanut allergy was the result of their body thinking birch (Bet v 1) was really peanut (Ara h 8). Evil birch can also cross-react with soybean (Gly m 4).

So...the takeaway is that there's a continuum of severity of reactions when it comes to these look-alike proteins. Some are just not likely to be problematic, but with others, whether a full-out allergic reaction occurs may depend on the individual's body chemistry, how much it "looks" like the other protein and how durable the protein itself is. Knowing which protein is involved is only the first step in the process and a food challenge is still the best way to figure out if an allergy exists.

So now the question is...which epitopes cause the problems in people who are allergic? And why would knowing which epitope help us in any way to understand our child's allergy? Can we use this technology to know which allergies are life-threatening? Persistent? Interlinked, so we can predict which foods not to give our kids? Or is it just another $300 we'll never see again?

Part 2 - Why Should We Care.


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