Monday, February 16, 2009

Understanding the Western Blot

Many patients ask me questions about the meaning of the Lyme Western Blot test. I will try to clear up the mystery. Admittedly, it is a bit complicated.

First of all, one must understand that the Western Blot test is used to detect antibody responses made by a host- in this case humans, exposed to the Lyme bacteria, Borrelia burdorferi. Antibodies are produced when special specialized circulating immune cells encounter proteins or other molecules- called antigens, on the surface of a foreign invader. This information is transmitted through a complex process to specialized cell which produce antibodies. Antibodies are tiny proteins- immunoglobulins, which attach to target areas found on the surface of the germ, Lyme in this case. Finding antibodies directed at a particular pathogen is like finding footprints in the sand. It says "Lyme was here," like a name etched on an old oak tree. It in no way proves that Lyme is still present in the body. This is key to understanding the divergent views held by the IDSA and ILADS.

Our immune system can produce several classes of immunoglobulins. For the purpose of evaluating exposure to Lyme only two are relevant: IgM and IgG.

If one looks at the the areas of the Lyme bacteria which are antigenic- the areas associated with antibody formation, one generally finds 14 different regions or molecules. If these proteins are allowed to diffuse on a specially prepared strip or Blot, one finds that these tiny antigentic areas separate out based on their respective weights. These Lyme related antigens have molecular weights which progress from 18 Kilodaltons (Kd) to 93 Kd. The unit of measurement is frequently omitted- frequently the bands are referred to only be a number, 41 for instance.

To perform a Western Blot test a patient's serum is incubated with a specially prepared Blots or strips containing the Lyme antigenic proteins. If antibodies are present in the blood/serum they cause a reaction on the strip. Anyone who has looked at a home pregnancy test or a rapid strep test is familiar with the appearance of such a reaction on a test strip. The reactions show up as linear streaks called bands. Individual bands can be identified by their position on the strip. When the lab performs a Western Blot test four strips are prepared. There is a Blot which detects IgM antibodies and a Blot which detects IgG antibodies. Both are matched against control strips.

It should be noted that the significance of IgM versus IgG antibodies may be very different regarding Lyme compared with many other infections. This topic will not be addressed here, but it is extensively discussed elsewhere.

Frequently results are referred to as CDC positive or negative. These designations are misleading. In 1994 the CDC established a national surveillance criteria for Lyme disease. A national standard for reporting a positive DIAGNOSTIC result has never been developed. Most labs erroneously only report results based on these CDC criteria. The CDC criteria was developed concurrently with the Lyme vaccine. Certain key bands were omitted in the CDC test because these bands would react in persons who had received the Lyme vaccine. For a variety or reasons, the vaccine was removed from the market. Unfortunately, some key bands were never added back to the most commonly used Lyme Western Blot tests.

Of 28 possible bands, the standard CDC test, which is not a diagnostic test, reports only 13 out of a possible 28 bands. Only 3 IgM bands are reported and only 10 IgG bands are reported. The test is called positive if 2/3 IgM bands react or if 5/10 IgG bands react. Unfortunately, the CDC has recently made the test even more restrictive. If a patient has a positive IgM response it must be followed by a positive IgG response after 4 weeks to be considered a positive result.
Again, it must be emphasized that this is a surveillance test- a research tool- it cannot be claimed to be a accurate test for proving exposure to Lyme disease.

Other laboratories have developed alternate Western Blot criteria. For example, IgeneX labs will report a test positive if a patient has 2 critical bands in the IgM or IgG subset.

There has been a longstanding concern that cross reactivity may occur with some bands. In other words, a prior infection with a non-Lyme germ may cause a reaction to occur at some of the bands. Because of this, the bands have been analyzed to determine which are very specific for Lyme infection. If a patient shows reactivity with these very specific bands, the likelihood of a false positive reaction is low.

Doctors vary in their interpretation of Western Blot bands.. For example, if a patient has a reaction at even a single key band, some physicians will consider this a positive result. There is even some disagreement about which bands are very specific. Bands 23,31,34,39, and 93 are considered very specific by most. The significance of the 18 and 41 bands is open to more controversy.

Most laboratories will only report a positive band if it reacts with an intensity very close to the control band. Bands with an intensity which exceeds the control may be designated by a series of pluses or a percentage greater than the control.

Only IgeneX reports bands as indeterminate. This indicates that the reaction is not zero but is does not meet the strict criteria to be called a positive reaction.

It should be kept in mind that many patients who have Lyme disease may have minimal or even no reaction on a Western Blot. Many Lyme patients are known to be "seronegative." Western Blot reports are always interpreted along side a physician's overall clinical assessment of a patient.

The Western Blot is a tool used by physicians who diagnose and treat Lyme disease. The results from this test do not provide answers to questions relating to: the persistence of Lyme infection, a particular course of therapy or a patient's prognosis. Western Blot results, whether they are positive, negative, equivocal or controversial, must always be interpreted along side other clinical information. Typically, IDSA doctors and ILADS doctors are likely to judge results differently based on individual biases.

I discourage patients from obtaining Lyme Western Blots without first consulting with a physician. These results need to be analyzed based on a physician's clinical assessment of a patient. A positive result does not guarantee that a patient has Lyme disease and a negative result does not guarantee that a patient does not have Lyme disease.

12 comments:

Avalon said...

Doc, in all seriousness, did you hear the story about the trained chimp who attacked it's owner and neighbor in CT yesterday (near my home). It sounds like a bad joke, but the chimp was recently diagnosed with Lyme, and the owner was quoted as saying that the chimp had been more aggressive and unpredictable since becoming ill! It had never been known to be aggressive prior to Lyme.

lyme4achange said...

I have had 2 specific questions in regards to the WB for a while for which I am having trouble getting an answer. Perhaps you might help.

Band 58, IgG, I understand is a heat shock protien antibody reaction. If I am correct about this, what other common conditions can cause this band?

Band 23, IgM, I have been told is a very specific band, as you state as well. I am curious as to what OSP of the BB for which it is specific, if you know.

Thanks.

Lyme report: Montgomery County, MD said...

Much has already been said about specific bands. Band 23 represents a reaction to an outer surface protein C. This protein is "down regulated" while Borrelia is in the tick gut. It becomes expressed when Bb enters the mammalian host. The 41 band represents the flagellin- the tail or the spirochete, and is always expressed. The outer surface protein A, which corresponds to band 31, is "up regulated" in the tick gut and becomes down regulated in the host. It is only expressed- again up regulated in the host after prolonged infection. Therefore, a positive 31 band has been associated with longstanding infection and be a marker for neuroborreliosis. The 34 band, outer surface protein B is also seen later in the course of the disease. Some have postulated that 58 and 66 bands are seen with co-infection. I have seen no evidence to support this hypothesis. These bands are of little value in and of themselves.

Lyme report: Montgomery County, MD said...

Lyme and tick borne illness have been associated with a cornucopia of psychiatric maladies and manifestations.

lyme4achange said...

That is interesting, thank you.

I thought it might have baan OSP C but I wasn't sure because some of the references I found were not what I would call reliable resources.

I find this interesting because in my last WB, that was taken about 3 months into treatment, I had a HUGE Band 23 igM responce and little else.

I'd had Lyme already, untreated, for about 20 years (Spring 1987 until Spring 2007 when I acciedentaly got diagnosed by a local urgent care doc in West Maryland when I was in for vertigo.)I have esophilia (sp?) for at least a decade that I know of and a few other things that indicate there is some hosing of my immune system after all this time anyway...

I don't know what my labcorp WB was 3 months prior, all I know about that is that it was enough to have me reported by the urgent care guy. I do know my ELISA was at about 3.

Some state that they see heat schock proteins in MS and other things. This is where I hit a road block. (I do mount a rather high respomce IgG to this band). Again, not real reliable sources.

Thanks again.

My ILads doc doesn't know what else to do at this point with me and so I'm trying to determine what I intend to do next. Knowledge, of course, is power.

kristylou said...

Doc,

I have been terribly sick for 9 years, I was first tested for Lyme when I first got sick. It was a false positive. Since then I have been diagnosed with traansverse myelitis, MS and many others. I had a positive ELisa so my Doc did the WB test which shoed a positive IGM of bands, 41,39,23. The igg was negative. Please help me!! I dont understand and neither did my Doc, she wants me to go to a Infectious Disease Doc, I would rather see a Lyme Literate Doc. Do you know of one in TN? or nearby... Does that WB mean I have Lyme? Thankyou so much I need answers!!

Murphy's Blog! said...

Kristylue,
I am in Nashville Nd can put you in touch with the right person. I have a similar story and I think I can help. My dog and I both went through the same thing and because of our friend who is very educated in tickborne illnesses, we are healthy now! Give me a call or email...
Tiffany (760) 402-4307
Buckeyevernon@yahoo.com

Murphy's Blog! said...
This comment has been removed by the author.
Barbara said...

In need help. I have been sick over a year with debilitating shoulder pain from side to side. Been to 17 doctors, and finally got a western blot. It came bacl present on IgG 41, IgG 23, and IgM 41...I am confused and noone thinks I have lyme. Do I have lyme for sure??If so,how do I tell if it is early or lat stages?/And why won't any doctors see that by my results??Can antibitiotics still help me??

Scott said...

My son and wife both have disseminated LD, not diagnosed for 18 months and 3 years, respectively.

Son has IgM 23 and 41, wife only has IgM 23, and neither have any IgG bands...

any thoughts?

Marc said...

We need your help, Doc!!

My fiancee has been struggling for several months with symptoms that SCREAM Lyme Disease. Two rheumatologists want to call it Fibro, but none have been willing to even TRY a simple 4-6 week course of Doxy, to see if there's any chance that it could be Lyme.

After several doctors, we finally found a PCP that was willing to at least do the TESTS. and of course, they followed CDC guidelines.

The ELISA test came back positive, but the Western Blot (Quest) came back CDC seronegative.

On this test printout there are two columns for each band. All of the IGG bands read "NONREACTIVE" in both columns.

One of the IGM rows, band 23, reads "REACTIVE A" in column one, and "NONREACTIVE" in column two.

Can ANY meaning be extracted from this, when combined with the presentation of practically EVERY Lyme symptom ever expressed by anyone, ever? This poor girl is SICK, and rarely has a good day anymore.

We've gone so far as to actually order the Doxy online ourselves (reputable overseas pharmacy, used by many others in our situation), and are prepared to start self-treatment for up to 62 days (200mg twice per day Doxy to start) just to see if there's even a mild improvement as some have reported (some, by three weeks "feeling great" with a Herx at week 4). We even ordered the Flagyl for the cyst form of the bacteria, but are more hesitant to try two at once.

Anyway, my question regarding the WB band 23 above is our primary concern. If it's NOT Lyme, we'll accept that, but there's been a specific exposure (we have photos of her Lyme rash), as well as precedent in our area (I've had it, my son has had it).

We don't know what to do... we just don't believe the Fibro diagnosis (yet?) and want to at least TRY... SOMETHING.

Your advice and comments are very much welcomed!!

Kristin said...

Hi, Just joined your blog post - interesting that you are in Montgomery Co. MD because my husband's family lives in Clarksburg, MD. And, if I have Lyme, as I was just diagnosed, then I suspect it came from a tick in MD since our dog would get the deer ticks on her nose when we would visit. My question is this: My practitioner says I am positive for Lyme. I did not have the ELISA. I had the Western Blot, which showed IgGP23 Present/Abnormal and IgG P58 Present Abnormal. All three IgM are absent. From what I've gleaned, the 2 present/abnormal does not constitute Lyme according to the CDC. I am preparing to go on antibiotics, and I already have UC which I think was exacerbated or brought on by 3 YEARS of antibiotics (1992-1995) for a disease I didn't have. I don't want to doubt my practitioner. And I certainly have had terrible arthralgia, etc for almost four years now. But I also want to make sure that I'm doing the right thing with this treatment, and I'm SO confused! Sometimes too much information seems worse. ANY advice is appreciated.