The reason for an ELISA first, then a western blot

Hi all, I'm new around here. I saw on another thread:

Pirouette said...
I am still searching for a good source that effectively explains or debunks the purpose for the sequence [of the two-tier test], instead of going straight to the more specific WB.

I have a PhD in biochemistry, which is the field that invented Western blots and ELISAs, so I can shed a little bit of light on this topic in a general sense.

It makes a lot of sense, from the perspective of a scientist, to screen samples with a high-throughout method such as ELISA where you can test 96 or 384 samples at a time. It's cheaper, easier and faster. Western blots are slow and only allow you to test a small number of samples. This is the sole reason that tests like the ELISA were developed, to make testing cheaper and faster.

I have used a similar method to screen a large number of samples in a 96-well plate (not an ELISA but another biochemistry technique). If a sample is positive, you investigate it further with another test; if it's negative, you discard it. I don't know if there's a name for this, but it's common in any high-throughput screening. Usually the second test reveals more detailed information and is more accurate, but slower and more expensive.

Scientists have to economize, just as doctors/insurance companies do. As a grad student running each test by hand, I couldn't have spent the time and money to thoroughly test every sample. So I used an initial test to pinpoint which ones were worth looking into.

The problem with this approach is, it relies on that initial test to be accurate and appropriately sensitive. I'm sure you guys are aware that the Lyme ELISA is notorious for giving false negatives. I suspect this is an issue of the sensitivity thresholds they use. (I can expand on this if for anyone who's curious.)

As scientists, we have to make some assumptions in order to be able to do anything. We try to test out assumptions and make sure they are correct. Somehow, something went very wrong with the assumptions they made when they developed the ELISA, and they are just sticking to their guns on it anyway.

Unfortunately for all of us patients, the doctors who interpret these tests usually don't remember enough of their biochemistry lab class to understand that the border between positive and negative is somewhat subjective. Some scientist had to draw a line and say "This much color is positive; below that is negative," with the awareness that some level of false negatives and positives are going to occur. Deciding where to draw that line is complicated, especially with any antibody-based method because they're prone to noise (color showing up for no good reason).

But doctors are trained to interpret tests as black-or-white, because its easier to teach and to apply in practice. They aren't really educated in which tests are more fuzzy. They wouldn't necessarily ever apply that information in real life anyway, since insurance companies enforce black-or-white standards.

Anyway, sorry I'm not citing any sources on this, it's one of those things that is such common knowledge within biochemistry that it's a little difficult to cite. I could try digging into historical literature on the ELISA if anyone really, really needs a source for this general practice. But sources specific to the development of the Lyme ELISA may not exist in the literature.

My Province (in Canada) uses the IFA for the screening tool. Mine was negative, so of course no WB was done.

A couple months later I tested Positive with Igenex IgM WB.

The thing is...when we're dealing with a devastating illness...they shouldn't be allowed to get away with NOT doing the WB....since the Elisa/IFA screening is not always accurate.

So, even though it's a cost- saver...that doesn't really explain why it's done when it's not reliable.
I can tell you that the money saved by not doing the WB is exceeded many times over by the costs of all the other tests/mri's/specialist appts. the patients are having when this comes back negative.
In the end it isn't saving money.

How do we get that across?

Thanks for sharing! Always good to have more scientists on the forums. I was actually going to ask for citations on the false negatives until I read the end of your post. If you find any or wish to expand on the sensitivity thresholds or anything related to false negativity I'd be very interested to hear your perspective in detail. My gut has always told me the tests are questionable but it's been a hard position to substantiate with the available literature.

Biscotta- I am so interested to ask you about your experience. Especially from a bio-chemist! I was recently diagnosed by an LLMD, but I am still skeptical.

I read one of your other posts about not improving from treatment.

Did you do a western blot? what were your bands?

I had igm 31 ind, 41 ind
igg 30+, 41+, 58++, 66+

igenex negative...

been treating for a couple months now. improving so far...

i hope you see this and appreciate your response.

Hi guys, thanks for the responses! I have been fortunate enough lately to be able to work part-time from home, so sorry for the delayed reply.

Psilociraptor -
Basically, what I mean about the sensitivity thresholds is it seems like they have set the bar for a positive too high. The high prevalence of false negatives makes me think they require quite a bit of color in order to call it a positive. However, there could be other issues, like Lyme patients' antibodies being low at certain stages of disease progression. I would have assumed this to be the case for late-stage Lyme but the paper Pirouette linked says that early stage has the same problem. (Thanks Pirouette for that study!) But honestly, I think the problem is more their sensitivity than our crappy antibodies.

Girlie -
That's great to hear that there's movement on this issue in Canada. I know that the US's IDSA is a huge barrier here so it would be great if other countries allow a little more room for alternate perspectives. And it's also great that you have a supportive GP!

Missouri -
I can't say I was never skeptical of the LLMDs, because as I've mentioned, they don't have the ability to back up their position with very strong clinical evidence. A *lot* of the reason that I decided to put my faith in them is because the community of Lyme patients advocate for them so strongly. I figured that if so many patients think that's the best way to go, there must be something to it. Once I got to know the LLMDs and their staff, I realized that they're still scientific in their approach, trying different combinations of drugs, different supplements, making observations, seeing what works. It's just that they can't publish a lot of that data.

But yes, I treated for about three years, and ultimately I am still left with some very severe symptoms. Some symptoms got better: brainfog, air hunger, balance problems, and on and on. I used to have so many weird symptoms.

Now, it's mostly fatigue and orthostatic intolerance. I can't be on my feet for very long, and even sitting straight upright is difficult. I'm able to use a laptop in bed, which is the only reason I can work. Each year, I'm able to do less and less. It's getting to the point where I'm losing the ability to keep myself fed: shopping for food, cooking meals, etc. I'm not making much money so I have to live with family anyway, and they're taking over a lot of those daily tasks.

It's really hard. I'll be posting soon to ask for help, because it's terrifying to imagine where I will be in 5 or 10 years.

Your WB is very interesting. You're actually one band shy of the elusive CDC diagnosis on your IgG.

My first WB was in 2011:
IgM 18+,31+,34+,41+
IgG 18+, 23-25+, 41++, 58+ and a few ind's

How long have you been ill? For people who have had Lyme in their system for a long time, their immune system weakens, and they produce less antibodies. My IgG reactivity went *way* down from 2011 to 2014. Interestingly, my IgM reactivity stayed about the same.

I'm glad to hear that you're improving, though. That's wonderful news, and to be honest, the best sign that you can hope to see. So much of what LLMDs do is just based on symptoms - try this and see if you get better, if not, then try that.

Pirouette -
I actually found you guys just through Google. I was looking into cistus tea and found an old thread on here. I'm looking into herbal self-treatment at this point, so I wanted to ask if you guys can point me in the right direction. (I haven't made that post yet but I will soon!)

Thanks for clarifying what you meant about the ELISA in the original quote. I definitely didn't intend for this to be a defense of the ELISA, really just an explanation of why it probably made sense at the time that it was developed. However, I do think that the sensitivity thresholds they use are off, and it seems to me that should have been apparent to them from the beginning.

But yes, I absolutely agree that it's indefensible for the CDC to continue to recommend the ELISA. It's also ridiculous that they continue to ignore the bands that were used in the failed vaccine, because those bands are highly specific for Lyme and should absolutely be used for diagnosis. But they just pull the "not for diagnosis, just for monitoring" card.

So this reveals a problem in our medical system - doctors regularly rely on a source for diagnotic criteria that is explicitly not intended to be used for diagnosis. I'm sure that they do so because it's the most authoritative, centralized source of information. It isn't realistic for the average PCP to go looking up diagnostic guidelines in the medical literature for every disease. However, unless the government created some other resource for diagnostic criteria, the only other source of criteria would be organizations like the IDSA.

I'm not a conspiracy theorist, but I am cynical as all hell. It seems to me that the way that the IDSA and CDC keep sticking to their guns on these issues has to do with money. But I don't know enough about all the things you listed in (C) to understand their motivations. The Lyme vaccine is deader than dead, and I'm assuming that any lawsuits from it have long been settled. So where is the incentive to stick to these criteria? Profits from running tons and tons of ELISAs? Wouldn't the patent have expired by now? Or dog vaccines? Same thing on the patent there too. I just don't get it. It just seems like there's a more complex situation going on that I know nothing about. It seems like you know a lot more about it than I do.

Post Edited (biscotta) : 10/11/2017 9:39:09 PM (GMT-6)

Missouri -
Thanks for the empathy! I also went two years from being bitten to diagnosis. In my case, I didn't realize it was a tick bite. I did have a rash but not a textbook bullseye rash, and my doctor dismissed it. I was asymptomatic for a year, and then spent another year trying to get a diagnosis.

about only having coinfections... Each tick can host different microorganisms, so it's possible in theory to have a tick carrying only coinfections. However, I don't think that's what's going on with you.

Each band on the western blot represents a protein from the Borrelia bacteria. Your antibodies are applied to those proteins. The lab technician then looks to see which proteins have antibodies stuck to them. Since antibodies bind pretty specifically, if you have antibodies sticking to those proteins, it's probably because your immune system has encountered those exact proteins.

However, there are some cases where antibodies can bind if you've encountered a very similar protein. Western blots can also give false positive bands because of non-specific binding (these tend to be "ind" though).

I would think that it would be statistically unlikely that you would have IgG antibodies to four Lyme proteins if you were not infected with Lyme disease at some point. There's probably a higher probability that your immune system isn't functioning so great after being infected for two years.

So, the Lyme WB won't tell you anything about any coinfections. It's specific to Lyme.

Missouri said...
Interesting you say that when almost everything I've read says those bands are non-specific to Lyme as in they cross react with other baddies. 41 syphilis, yaws, etc. Can't find much on band 30. I've read in a lot of places 58 and 66 cross react with other baddies too. hmmmm...

Your Igg results:

igg 30+, 41+, 58++, 66+

From Igenex website:

CDC criteria: 5 of the following 10 bands are present: 18, 23-25, 28, 30, 39, and 41, 45, 58, 66 and 83-93kDa.

Antibodies are typically low in early illness because it takes about 2-4 weeks to produce. This would actually be pretty general to all illnesses. The controversy is more in late stage. I've seen one paper suggest that immune complexes can lead to false negativity https://www.ncbi.nlm.nih.gov/pubmed/1967770. Borrelia is also known to cause humoral dysfunction but I don't know the implications of that for testing. Another red flag to me is that regional variation of borrelia s.l. species has been poorly explored. As well as the fact that cultured isolates produce different antigen than live infection. It's very likely in my mind that the antigen pool used for the tests is not reflective of all real world scenarios. Also, just out of common sense I have to imagine that any parasitic illness, with it's immunosuppressive nature, would lead to variable outcomes that preclude a one size fits all diagnostic scheme. I mean, we're talking about the human as an ecosystem where each interaction triggers a nonlinear cascade of consequences defined by innumerable variables. How we ever got the idea that diseases, treatments, diagnostics, etc could be standardized for anything other than scientific purposes is beyond me.

As for sensitivity thresholds that makes sense. I've read WB can be very subjective when it comes to deciding which bands are strong enough. Do you know if they use pixel counting for this? Or just eyeball it? I would think the subjectivity would be greatly reduced by eliminating technician bias. In either case I suppose any standard threshold can be subject to bias itself.

Biscotta, I see you're interested in an herbal approach. Coming from a biochem background myself I was super impressed with Stephen Harrod Buhners 2015 book Healing Lyme. I mean, it's quite clear he's an herbalist and not a scientist. But for the most part he's done his research better than most, presents it coherently, and gives an expansive list of herbal suggestions for each and every little thing. There's nothing woo woo about his material. He's a really grounded fellow. I've been using his protocol exclusively. No doubt with some bumps in the road, but it certainly is potent and I have talked to many people in full remission or partial remission from it. Just gotta keep tinkering with it. But already I've experienced some good benefit. I don't think you can do better. Not to say there aren't herbs he doesn't recommend that might be good. But when you're looking for a starting place Buhner is really good. He got me totally out of reductionist mentality of "this is a bacteria, we must kill it with antibiotics" and into the "whole plants are chalk full of anti inflammatories, quorum sensing inhibitors, adhesin inhibitors, efflux pump inhibitors, biofilm degrading comprounds, immune modulators, secretion system inhibitors, etc" mindset. Treating non linear diseases with non linear therapies. Herbs really embody holism. And yet, my education never exposed me to herbs as anything other than a nuisance of organic matter that novel drugs were trapped inside. Patiently waiting for scientist to tediously chip away all the undesirables to make them viable. It's kind of funny looking back. What a bizarre world we live in.

On the topic of cross reactivity. One thing to keep in mind is that many surface antigens play a role in cellular adhesion. So pathogens with similar tissue tropisms actually have a potentially strong likelihood towards immunological cross reactivity even if they are unrelated. This is because the charge and shape of these proteins is specifically evolved to bind adhesive proteins in the human tissue meaning they can generate a similar antibody response. What Biscotta is saying about the "simplest explanation" is still true. Very few pathogens occupy the exact same niche so while there may be overlap, the more Lyme positive bands you have, the less likely it is to be something else. But all in all I don't think it matters THAT much. It's always comforting to be able to point to something and say "Hey that's Lyme disease". But EVERY chronic infection is going to be complicated by coinfection with transmitted illnesses, latent viruses, and/or commensal organisms. Every person is going to have to personalize treatment approach through trial and error. So ultimately it doesn't matter too much whether this is Lyme disease, or syphilis, or some yet unidentified protozoan. What really matters is that you're scrapping the idea of chronic illness as something other than chronic infection. And you're scrapping the idea of chronic infection as something linear that can be treated with linear approaches. You're looking at the human as a complex ecosystem of diverse cellular bodies and treating it in that light. I think that's really more important than identifying all the players involved. Because, face it, there will always be a limit on our understanding of the details. We have yet to apply metagenomics clinically and even when we do it's a long way from understanding all the subtle interactions between microorganisms present. Which ones are more relevant, which ones are just supporting players, which ones are totally benign, etc. Another reason i really like herbs. I don't have to target every microorganism with laser precision. I can just assume that I get pretty good coverage with plants. Though more knowledge is always helpful.

"but I am cynical as all hell."

:O Are we finally allowed to swear on these boards???? Testing **** crap darn it

Edit: swear fail. oh well. at least I can say "hell"

Post Edited (Psilociraptor) : 10/13/2017 7:34:13 AM (GMT-6)

Missouri - No problem!

Psilo -
Great point on the variations of borrelia species as well as the uncountable variables within the human body.

Yeah, you only eyeball a WB if you're like I was in grad school, "Is there any protein there? Y/N" My experiments were just qualitative. However other members of my lab would do semi-quantitative WBs with a scanner and a program that measures the intensity of the color and the size of the bands. I'm sure that any lab that's using WBs for diagnostics would use software rather than eyeballing. Igenex probably has a standard definition for how much intensity is "ind", how much is +, how much is ++.

But yes, deciding where to make those cutoffs is tricky. I like that they at least have an "indeterminate" result, it allows for a shade of grey in between the black and white.

Thanks for the recommendation of Buhner, it sounds like exactly what I need to get started. I ordered his book. One thing I'm wondering about is if I will need help to decide whether I need should target any coinfections. I've long felt that my LLMDs were just missing a piece, because they were able to resolve many symptoms. We mostly treated for Lyme and babesia but I think both LLMDs did use things that could hit multiple coinfections.

I hear you about the reductionist thinking, though! My lab did a lot of work with natural products. In that field, it's like the whole goal in life is to figure out what one molecule is the magic bullet. I remember being frustrated that nobody was doing that kind of work on some of the more successful Lyme herbs.

But you're right, there's no need to reduce it down to the most potent molecule, because there are plenty of other beneficial components in these herbs.

Also an excellent point about cellular adhesion.

I was thinking more about what the angle could possibly be for the IDSA and I'm thinking liabilty. Can you imagine if a class-action lawsuit could get traction for every ELISA-negative chronic Lyme sufferer? It's like when an industrial company gets caught polluting an area and making people sick, they don't just fess up. They lawyer up. But at least industrial companies don't control the means of diagnosis. A cancer cluster is a cancer cluster, it's pretty strong evidence.

Anyway, that's all pure speculation. But the intractability of the situation makes me feel like we won't get real movement in the US until all the current IDSA guys are long gone.

Biscotti - Great post and welcome! I love all the science. I had 7 ELISA, all negative, told it was in my head, etc. 6 years of bouncing around from Neuros, Cardiologists, Endocrinologists, a bushel of Xanax and Klonopin in the garage. My LLMD ordered the Igenix and Igenix said on my results that my Lyme was considered positive by Igenix but negative by CDC standards. I guess I had the band they throw away. I tell every doctor I know, including the ones who missed my Lyme to go straight to Western Blot and do it after a two week Doxy run. Whether it will catch on, who knows, but if I can save one person....I actually went back to the Neurologist (head of Neurology, famous Hospital) and told him I was getting treated for Lyme and he said, "Well, sometimes placebos help." This guy is in THE position to actually ruin people's lives with that attitude. What a MFkr!!!!

I hope you find some relief and that you consider the Buhner herbs as suggested. I have used most of them at various points because the co-infections become somewhat of a whack-a-mole situation. Plus the added benefit of lowering systemic inflammation makes life much more bearable. Although, if you are bed ridden, you might need IV antibiotics. I've seen them turn people around.

Please also delve in the the world of MTHFR. Many people who get these chronic illnesses have trouble detoxing and need to figure out how to increase Methylation pathways safely so they can detox effectively.

Psilo - I definitely think that the future of medicine is analyzing the gut microbiome and genetics to maximize health and healing. Actually, it's already here. This last round of Rifampin and all of my allergies are flaring. I'm pretty sure the Gut micro biome is not too happy right now and it's all connected. My spelling is a mess. Sorry.

Psilo - I just listened to an interesting podcast where the good bacteria (if they are happy and functioning well in large numbers) actually get the bad bacteria (what we call pathogens) to fall in line and start behaving. Apparently certain strains of Bacillus are the ring leader of the gut and send out electronic messages to try and influence behavior.

I'm a big Chinese Medicine person and I don't subscribe to the the body parts approach to healing. It's all one system that has to work together. However, the brain can tip the power scale if it's under stress and not thinking positively. Stress releases inflammatory cytokines that affect the balance of good and bad bacteria. It's all so fascinating.

I definitely sleep better when I take my probiotic before bed...it can't be a coincidence.