posted
I still need help in understanding the western blot test done for lyme. Based on the following lab results:
IGG by Western Blot P41 Present All other bands say absent Lyme IgG WB Interp. NEG
IgM by Western Blot P41 Present P39 Present P23 Absent Lyme IgM WB Interp. POS
Then, at the bottom of the page is says:
Lyme IgM/IgM AB <0.91 Index 0.00 - 0.90 Negative <0.91 Equivocal 0.91- 1.09 Positive >1.09
This test was done by Lab Corp.
Also, could testing positive for Epstein Barr Virus last summer have any affect on the Western Blot test results. Could the antibodies from Epstein Barr influence the antibodies for Lyme??
Pleaaaaaaaaaaaaase help, I am so confused?
Posts: 11 | From Maryland | Registered: May 2007
| IP: Logged |
Michelle M
Frequent Contributor (1K+ posts)
Member # 7200
posted
My GOODNESS.
LabCorp found lyme in a person! Get outta here!
You are quite lucky!
Your IgM test is positive. Band 41 can cross-react with other pathogens but Band 39 being positive is pretty good confirmation.
Not only that, but LabCorp doesn't even bother testing for two of the most specific bands of all -- I believe bands 31 and 34. Those might be positive as well but you'd never know with them. They and other labs pretend they have to omit those bands just in case you had the lyme vaccine.
No matter. You have a positive test.
The bottom portion looks like an ELISA test, which is negative. You're under their cut-off.
If your doctor tries to tell you that you don't have lyme because the ELISA is negative and therefore the western blot must be a "false positive," please run away fast.
I would suggest you find an LLMD! Post in the "Seeking a Doctor" section. Mainstream docs tend to either deny lyme completely or to undertreat it. You also need to be tested for co-infections at a GOOD lab, not Lab Corp.
Good luck!
Michelle
Posts: 3193 | From Northern California | Registered: Apr 2005
| IP: Logged |
CaliforniaLyme
Frequent Contributor (5K+ posts)
Member # 7136
Explanation of the Lyme Disease Western Blot by Carl Brenner
Inquiries about various issues relating to Western blot (WB) testing are frequently posted to the Lyme disease discussion groups on the Internet. Among the most commonly asked questions are: What laboratory techniques are used to carry out the assay? What exactly is being measured? What is a "band"? How are the results interpreted? What are the CDC criteria for a "positive" test? Although some of then medical jargon associated with immunology can be a little overwhelming, the scientific principles behind these tests are not difficult to grasp. The following article is offered as a primer in the techniques and interpretation of Western blotting, and should help most patients navigate their way through some of the medical and scientific terminology associated with the assay.
First of all, it should be noted that the Western blot is usually performed as a follow-up to an ELISA test, which is the most commonly employed initial test for Lyme disease. "ELISA" is an acronym for "enzyme-linked immunosorbent assay." There are ELISA tests and Western blots for many infectious agents; for example, the usual testing regime for HIV is also an initial ELISA followed by a confirmatory Western blot.
Both the ELISA and the Western blot are "indirect" tests -- that is, they measure the immune system's response to an infectious agent rather than looking for components of the agent itself. In a Lyme disease ELISA, antigens (proteins that evoke an immune response in humans) from Borrelia burgdorferi (Bb) are fixed to a solid-phase medium and incubated with diluted preparations of the patient's serum. If antibodies to the organism are present in the patient's blood, they will bind to the antigen. These bound antibodies can then be detected when a second solution, which contains antibodies to human antibodies, is added to the preparation. Linked to these second antibodies is an enzyme which changes color when a certain chemical is added to the mix.
Although the methodology is somewhat complicated, the basic principle is simple: the test looks for antibodies in the patient's serum that react to the antigens present in Borrelia burgdorferi. If such antibodies exist in the patient's blood, that is an indication that the patient has been previously exposed to B. burgdorferi.
However, many different species of bacteria can share common proteins. Most Lyme disease ELISA's use sonicated whole Borrelia burgdorferi -- that is, they take a bunch of B. burgdorferi cells and break them down with high frequency sound waves, then use the resulting smear as the antigen in the test. It is possible that a given patient's serum can react with the B. burgdorferi preparation even if the patient hasn't been exposed to Bb, perhaps because Bb shares proteins with another infectious agent that the patient's immune system *has* encountered. For example, some patients with periodontal disease, which is sometimes associated with an oral spirochete, might test positive on a Lyme ELISA, because their sera will react to components of Bb (like the flagellar protein, which is shared by many spirochetes) even though they themselves have never been infected with Bb. Therefore, some positive Lyme disease ELISA results can be "false" positives.
To distinguish the false positives from the true positives, a more specific laboratory technique, known as immunoblotting, is used. (The Western blot, which identifies specific antibody proteins, is but one kind of immunoblot; there is also a Northern blot, which separates and identifies RNA fragments, and a Southern blot, which does the same for DNA sequences.) In a Western blot, the testing laboratory looks for antibodies directed against a wide range of Bb proteins. This is done by first disrupting Bb cells with an electrical current and then "blotting" the separated proteins onto a paper or nylon sheet. The current causes the proteins to separate according to their particle weights, measured in kilodaltons (kDa). From here on, the procedure is similar to the ELISA -- the various Bb antigens are exposed to the patient's serum, and reactivity is measured the same way (by linking an enzyme to a second antibody that reacts to the human antibodies). If the patient has antibody to a specific Bb protein, a "band" will form at a specific place on the immunoblot. For example, if a patient has antibody directed against outer surface protein A (OspA) of Bb, there will be a WB band at 31 kDa. By looking at the band pattern of patient's WB results, the lab can determine if the patient's immune response is specific for Bb.
Here's where all the problems come in. Until recently, there has never been an agreed-upon standard for what constitutes a positive WB. Different laboratories have used different antigen preparations (say, different strains of Bb) to run the test and have also interpreted results differently. Some required a certain number of bands to constitute a positive result, others might require more or fewer. Some felt that certain bands should be given more priority than others. In late 1994, the Centers for Disease Control and Prevention (CDC) convened a meeting in Dearborn, Michigan [1] in an attempt to get everybody on the same page, so that there would be some consistency from lab to lab in the methodology and reporting of Western blot results.
Before we get to the recommendations that resulted from this meeting, we need to understand one more facet of the human immune response. Many patients have noticed that their Western blot report usually contains two parts: IgM and IgG. These are immunoglobulins (antibody proteins) produced by the immune system to fight infection. IgM is produced fairly early in the course of an infection, while IgG response comes later. Some patients might already have an IgM response at the time of the EM rash; IgG response, according to the traditional model, tends to start several weeks after infection and peak months or even years later. In some patients, the IgM response can remain elevated; in others it might decline, regardless of whether or not treatment is successful. Similarly, IgG response can remain strong or decline with time, again regardless of treatment. Most WB results report separate IgM and IgG band patterns and the criteria for a positive result are different for the two immunoglobulins.
Finally, in setting up a nationwide standard for a positive WB, one makes several assumptions -- that all strains of Bb will provoke similar immune responses in all patients, that all patients will mount a measurable immune response when exposed to Bb, and that the IgG immune response will persist in an infected patient. Unfortunately, none of these is always true. Therefore, a judicious interpretation of Western blot results in a clinical setting should take into account both the vagaries of the human immune response and the possibility that strain variations in Bb might produce unusual banding patterns.
The CDC criteria for a positive WB are as follows:
* For IgM, 2 of the following three bands: OspC (22-25), 39 and 41. * For IgG, 5 of the following ten bands: 18, OspC (22-25), 28, 30, 39, 41, 45, 58, 66 and 93.
How were these recommendations arrived at? The IgG criteria were taken pretty much unchanged from a 1993 paper by Dressler, Whalen, Reinhardt and Steere [2]. In this study, the authors performed immunoblots on several dozen patients with well characterized Lyme disease and a strong antibody response and looked at the resulting blot patterns. By doing some fairly involved statistical analysis, they could determine which bands showed up most often and which best distinguished LD patients from control subjects who did not have LD. They found that by requiring 5 of the 10 bands listed, they could make the results the most specific, in their view, without sacrificing too much sensitivity. ("Sensitivity" means the ability of the test to detect patients who have the disease, "specificity" means the ability of the test to exclude those who don't. Usually, an increase in one of these measures means a decrease in the other.)
The IgM criteria were determined in much the same fashion (by different authors in different papers). Fewer bands are required here because the immune response is less mature at this point.
Several studies have shown that the first band to show up on a Lyme disease patient's IgM blot is usually the one at 41 kDa, followed by the OspC band and/or the one at 39. The OspC and 39 kDa band are highly specific for Bb, while the 41 kDa band isn't. That's why the 41 by itself isn't considered adequate.
Here's the rub, though: the CDC doesn't want the IgM criteria being used for any patient that has been sick for more than about six weeks. The thinking here is that by this time an IgG response should have kicked in and the IgM criteria, because they require fewer bands, are not appropriate for patients with later disease.
A number of criticisms have been offered of the CDC criteria since their adoption in 1994. The first is centered on the CDC's failure to make any qualitative distinction among the various bands that can show up on a patient's Western blot. A number of Lyme disease researchers feel that different bands on a WB have different relative importance -- that "all bands are not created equal." For example, many patients with Lyme disease will show reactive bands at, say, 60 and/or 66 kDa. However, these correspond to common proteins in many bacteria, not just Borrelia burgdorferi, and so are of limited diagnostic usefulness, especially in the absence of other, more species- specific bands. The band at 41 kDa corresponds to Bb's flagella (the whip like organelles used for locomotion -- Bb has several) and is one of the earliest to show up on the Western blots of Lyme disease patients. But for some reason it is also the most commonly appearing band in control subjects. This may be due to the fact that many people are exposed to spirochetes at some time in their lives and so their sera might cross react with this protein.
On the other hand, certain other bands are considered highly specific for Bb -- the aforementioned 31 kDa band, for example, or 34 (OspB) or 39 or OspC (anywhere between 22 and 25). The 83 and 94 kDa bands are also thought to be species-specific. Many Lyme disease scientists believe that any patient whose IgG Western blot exhibits bands at, say, any three (or even two) of these locations almost certainly has Lyme disease, regardless of whether or not any other bands are present. They feel that these bands on a Lyme Western blot are simply more meaningful than other, less specific ones and that a rational interpretation of a WB result should take this into account. Unfortunately, this does not often happen, and will happen even less with the new CDC criteria.
A second criticism of the CDC Western blot criteria is that they fail to include the 31 and 34 kDa bands. This does indeed seem like an odd decision, since antibodies with these molecular weights correspond to the OspA and OspB proteins of B. burgdorferi, which are considered to be among the most species- specific proteins of the organism. So why didn't Dressler et al. include them? Answer: These bands tend to appear late if at all in Lyme disease patients, and did not show up with great frequency in the patients that the Dressler et al. group studied (though they did show up sometimes). As a result, they weren't deemed to have much diagnostic value and didn't find their way onto the CDC hot list. However, while the absence of either of these bands from a patient's immunoblot result does not rule out Lyme disease, their presence is hardly meaningless. Thus, many Lyme disease experts believe it is a serious mistake to exclude these two antibody proteins from the list of significant bands. The CDC's decision to do so seems particularly strange in light of the fact that it is the OspA component of Bb that is being used as the stimulating antigen in the ongoing experimental Lyme disease vaccine trials. As one immunologist remarked shortly after the 1994 CDC conference, "If OspA is so unimportant, then why the heck are we vaccinating people with it?"
Finally, it is important to keep in mind that no matter how carefully the Western blot test is carried out and interpreted, its usefulness, like that of all tests that measure B. burgdorferi antibodies, is ultimately contingent on the reliability of the human immune response as an indicator of exposure to B. burgdorferi. There are several scenarios in which the lack of a detectable antibody response may falsely suggest a lack of B. burgdorferi infection.
First, it is well established that early subcurative treatment of Lyme disease can abrogate the human immune response to B. burgdorferi [3]. Although this is not thought to be a common phenomenon, a recent comparative trial for the treatment of erythema migrans found that a majority of patients who failed early treatment and suffered clinical relapse were seronegative at the time of relapse [4]. Even treatment for disseminated Lyme disease, in which the patient's IgG immune response was previously well-established, can render a patient seronegative after treatment despite post- treatment culture-positivity for B. burgdorferi [5,6].
In addition, patients with Lyme disease may not test positive for exposure to B. burgdorferi because their antibodies to the organism are bound up in immune complexes [7]. Once steps are taken to dissociate these immune complexes, free antibody can be detected; however, this is not routinely done when performing serologic tests for Lyme disease. Finally, an indeterminate number of patients with late Lyme disease are simply seronegative for unknown reasons [8]. The actual percentage of such cases as a proportion of all Lyme disease cases is impossible to estimate, since most studies of late Lyme disease enroll only seropositive patients, which tends to reinforce the circular and erroneous notion that virtually all patients with late Lyme disease are seropositive.
It should also be noted that a positive Western blot is not necessarily an indication of active Lyme disease. A patient's immune response to B. burgdorferi can remain intact long after curative treatment for a Lyme infection; therefore, the results of a Western blot assay should always be interpreted in the context of the total clinical picture.
REFERENCES
[1] Proceedings of the Second National Conference on Serologic Diagnosis of Lyme Disease, October 27-29, 1994.
[2] Dressler F, Whalen JA, Reinhardt BN, Steere AC. Western blotting in the serodiagnosis of Lyme disease. J Infect Dis 1993;167:392-400.
[3] Dattwyler RJ, Volkman DJ, Luft BJ et al. Seronegative Lyme disease: dissociation of specific T- and B-lymphocyte responses to Borrelia burgdorferi . N Engl J Med 1988;319:1441-6.
[4] Luft BJ, Dattwyler RJ, Johnson RC et al. Azithromycin compared with amoxicillin in the treatment of erythema migrans. Ann Intern Med 1996;124:785-91.
[5] H upl T, Hahn G, Rittig M, et al. Persistence of Borrelia burgdorferi in ligamentous tissue from a patient with chronic Lyme borreliosis. Arth Rheum 1993;36:1621-6.
[6] Preac-Mursic V, Marget W, Busch U, Pleterski Rigler D, Hagl S. Kill kinetics of Borrelia burgdorferi and bacterial findings in relation to the treatment of Lyme borreliosis. Infection 1996;24:9-18.
[7] Schutzer SE, Coyle PK, Belman AL, et al. Sequestration of antibody to Borrelia burgdorferi in immune complexes in seronegative Lyme disease. Lancet 1990;335:312-5.
[8] Liegner KB. Lyme disease and the clinical spectrum of antibiotic responsive chronic meningoencephalomyelitides. (Abstract, 1996 LDF Conference, Boston. MA)
Indirect Tests (serum antibody tests): ELISA; Western Blot; IFA; Borreliacidal Antibody Assay (Gunderson test); T-cell Activation Test Direct detection tests: PCR (DNA amplification); Lyme Urine Antigen Test (LUAT); Antigen Capture Test; culturing of skin, blood, CSF, urine, or tissue; immune complex / antigen-antibody test Tissue Biopsy and Staining: Silver Stain; Gold Stain; Fluorescent Tagged Monoclonal Antibody Stains; Acrodine Orange; Gram Stain; Muramidase; etc. There is a great deal of confusion and controversy surrounding Lyme disease testing. The first problem is that most of the manufacturers of these tests want you to believe that their tests are the best. At every medical convention, I listen to sales pitch after sales pitch from sales people making their product sound infallible. Often the terminology is confusing and the customer frequently misinterprets what is really being said.
For example, a salesman may say the rate of false positive or false negative is less than one percent. This sounds like the test is more than 99% accurate. In reality, what it is saying is if you have 1000 test samples from the same known laboratory sample, then in less than ten samples will there be a result that differs significantly from the other 990.
In any of this, did you hear the words: "percent reliability" or "percent accuracy" in diagnosing Lyme disease in humans? No! People often mistake "false positive rate" for accuracy. The truth is that no Lyme disease test to date is close to 100% accurate, because each test has its own particular set of shortcomings. So, while the first problem with Lyme disease tests is in the way they are promoted, the second problem is the way the tests are primed to recognize laboratory strains of Bb, rather than wild types. Third, the Lyme spirochete can hide in the human body, and fool the immune system into thinking it isn't there. So, no antibodies are produced, resulting in negative tests. Stealth technology isn't new, it evolved millions of years ago by the first bacteria that evaded its host's defenses.
Immune Responses
The first antibody our body makes in response to a foreign invader is usually immunoglobulin type M, abbreviated as IgM. This large antibody takes two to four weeks to be made in quantities large enough to be consistently measured. It is at its peak of production four weeks after exposure to an antigen. The IgM antibody will only stay in circulation for about six months, and then levels are usually too low to detect. If infection persists, this antibody may also persist. In general, a Lyme patient who consistently has detectable IgM levels is usually chronically ill, but its absence is not a reliable indicator of cure.
The second antibody we make after the IgM is the IgG antibody. This antibody takes four to eight weeks to form, and is gone in less than twelve months. It peaks at about six weeks. This antibody crosses the placenta, so an infected mother can pass this antibody to her child. An IgG antibody titer in a newborn does not have to mean active infection. It does mean the mother has had exposure, and the child must be carefully monitored for signs of the disease.
Because of the difference in the two antibodies, two separate tests are available to test for their presence. Therefore, a physician must specify whether or not a patient should have an IgM or IgG Western Blot, or an IgM or IgG ELISA test.
IgM:
This is the earliest of the antibodies to appear in response to an infection. It is produced in quantity. It is six times larger than the IgG antibody. Because of its size, this immunoglobulin does not cross the placenta. Since it cannot enter the fetus from the mother, any newborn that starts to make IgM antibodies against Lyme disease must be infected. However, a fetus exposed to Borrelia burgdorferi early in the pregnancy may never make an antibody response to the Lyme bacteria because the baby's immune system doesn't recognize it as foreign.
IgG:
This antibody remains the longest and is the foot soldier of the immune system. It attacks viruses, bacteria, yeast, toxins, and transplants. The IgG antibody can kill bacteria indirectly by tagging or marking the foreign invaders for destruction by the killer cells (T-cells, macrophage). Or, it can kill the bacteria directly by evoking compliment, a series of enzymes and proteins that will dissolve the intruder.
Note: It was once thought that plasma cells could produce antibodies that could conform to any shape necessary to attack foreign intruders. If this were true, we would have almost unlimited immunity. It is now thought that each person has a finite collection of specialized lymphocytes that are able to create a finite number of antibodies. Each antibody shape is predetermined, and can be produced by only one type of lymphocyte. When the body is invaded by a foreign antigen, it will stimulate one of these cells, and only that cell will begin to clone itself. This process takes several weeks. If we lack the right cell type to do the job, we are left with a gap in our immunity. This might account for why some Lyme patients with certain tissue types have greater morbidity, while others have relatively mild symptoms.
Dr. Alan Steere, M.D., observed that Lyme arthritis patients with tissue type HLA-DR2 and HLA-DR4 had more severe arthritis and chronic disease. Other tissue types have been associated with an increased incidence of multiple sclerosis and other neurological diseases. It might be that different patient tissue types might account for a difference in patient's symptoms to a greater degree than different strains of the bacteria.
It is known that this bacteria has an affinity for specific tissues. If you have a specific lack of immunity, this may cause the disease to manifest differently in those tissues. For example, let's say hypothetically that your heart is infected with Borrelia burgdorferi bacteria. Perhaps most people make an antibody that suppresses attachment of Bb to certain fibers in the heart. If you lack that antibody, the infection may continue more aggressively and manifest differently - for instance, causing an enlargement of the muscle fibers or destruction of the conduction pathways.
Instead of lacking a specific antibody, perhaps some individuals make a different kind of antibody, an antibody that not only attacks the bacteria - but may attack the heart as well! It is well known and documented that some patients produce auto- antibodies, which are antibodies that our own body produces that attack our own tissues. This is the basis of autoimmune disease. In some Lyme disease patients, an auto-antibody against cardiolipin has been clearly established in Lyme patients with Lyme carditis.
Perhaps, in addition to other Lyme tests, we should also be tissue typing patients and searching for auto-antibodies? Tissue typing requires a small blood sample, and costs about $200.
The Western Blot essentially makes a map of the different antibodies the immune system produces to the bacteria. The map separates the antibodies by the weight of their respective antigens and are reported in units called kilo daltons or kDa. For example, a Western Blot may report bands at 22, 23, 25, 31, 34, 39, and 41 kDa. Each of these bands represents an antibody response to a specific protein found on the spirochete. The 41 band indicates an antibody to the flagella 41 kDa protein and is nonspecific. The 31 kDa band represents the OSPA protein and is specific for just a few species of Borrelia, as is the 34 band OSPB, and 23 kDa OSPC.
In 1994, the Association of State and Territorial Public Health Laboratory Directors, under a CDC grant, decided that there should be consistency between labs reporting Lyme disease Western Blots, and that a specific reporting criteria should be established. The consensus committe, chaired by Dr. Michael Osterholm, Ph.D., MN, set nationwide standards for Western Blot reporting. This sounds good, but one could argue they made a bad situation worse. Prior to the hearing, virtually every lab had accepted bands 22, 23, 25, 31, and 34 kDa as specific and significant, and reported them as positive for exposure to Borrelia burgdorferi. Not only are these bands specific for Borrelia species, but they represent all of the major outer surface proteins being used to develop the Lyme vaccines. The committee, without any clear reasoning, disqualified those bands as even being reportable.
After the consensus meeting, those bands were no longer acceptable. The result was that what had been a fair-to-good test for detecting Lyme disease had now become poor, arguably useless. Many scientists have questioned these new reporting criteria, and several wrote letters of protest to both the committee and to laboratory journals. Many labs stopped reporting the actual bands and instead, simply reported the test as positive or negative, thus preventing any further interpretations. (90)
How badly did the Lab Directors bootstrap this test? The following is an analysis of the new guidelines presented as an abstract and lecture at the 1995 Rheumatology Conference in Texas, chaired by Dr. Alan Steere, MD. (1995 Rheumatology Symposia Abstract #1254, Dr. Paul Fawcett, et al.)
This was a study designed to test the recently proposed changes to Western Blot interpretation by the Second National Conference on Serological Testing for Lyme Disease, sponsored by the CDC. The committee proposed limiting the bands that could be reported in a Western Blot for diagnosis of Lyme disease. Out of a possible 25 bands, 10 specific bands were selected as being reportable. An lgG Western Blot must have five or more of these bands: 18, 21,28, 30, 39, 41,,45, 58, 66 and 93 kDa. An lgM Western Blot must have two or more of the following three bands: 23, 39, 41.
Conspicuously absent are the most important bands, 22, 23, 25, 31, and 34, which include OSPA, OSP-B and OSP-C antigens - the three most widely accepted and recognized Bb antigens. These antigens were the antigens chosen for human vaccine trials. This abstract showed that, under the old criteria, all of 66 pediatric patients with a history of a tick bite and bull's-eye rash who were symptomatic were accepted as positive under the old Western Blot interpretation.
Under the newly proposed criteria, only 20 were now considered positive. (The number of false positives under both criteria was zero percent.) That means 46 children who were all symptomatic would probably be denied treatment! That's a success rate of only 31%.
* Note: A misconception about Western Blots is that they have as many false positives as false negatives. This is not true. False positives based on species specific bands are rare.
The conclusion of the researchers was: "the proposed Western Blot reporting criteria are grossly inadequate, because it excluded 69% of the infected children."
Elisa Test
The Enzyme-Linked Immunosorbant Serum Assay, is the simplest, least expensive, easiest to perform, and most common Lyme test ordered. It is a test based on detecting the antibodies that our bodies make in response to being exposed to Borrelia burgdorferi (Bb). It is a preferred test by laboratories, not because it is more accurate than other Lyme tests, but because it is automated. Many different patient samples can be performed by a single machine simultaneously. This allows for a faster turnover, less costs, and theoretically, standardized test results that are consistent from lab to lab.
We are told by manufacturers, health departments and clinics that the Lyme ELISA tests are good, useful tests, but in two blinded studies that tested laboratories for accuracy, they failed miserably. Lorie Bakken, MS/MPH, showed in her studies that there was not only inaccuracy and inconsistency between competing laboratories, but also between identical triple samples sent to the same lab. In other words, identical samples often resulted in different results! In the first study, forty- five labs correctly identified the samples only 55% of the time.
In the latest study by the College of American Pathologists, 516 labs were tested. The overall result was terrible! There were almost equal numbers of false positives as false negatives. Overall, the labs were 55% inaccurate. The labs could only give a correct result 45% of the time. You are actually better off to flip a coin!
The basis of the ELISA test is that it can be primed to be very specific for particular antibodies. This is done by taking a laboratory sample of the Lyme bacteria and breaking the sample down into fragments. These fragments, or antigens, are then embedded on the side of a reagent vessel like a test tube. Then the patient's serum is added, and any free (non-complexed) antibodies specific for the test strain will then bind to the antigens, which are linked to special enzymes that will change color when antibodies are present. The sample is continually diluted until the reaction no longer occurs and no color change can be detected. The sample is then reported as a dilution ratio, such as one part serum to 256 parts water, or 1:256.
The ELISA test sounds simple and straight forward, but it has a couple of major flaws. Borrelia species are some of the most polymorphic bacteria known to exist. In other words, most Borrelia species can significantly change its surface proteins enough during cell division as to evade our immune system, and may differ from laboratory strains enough to result in negative tests, even if antiBb antibodies are present! In Europe, this problem is intensified because they have recognized three species of Borrelia that cause Lyme disease, and so they have available three separate ELISA tests. The questions in America are: 1) Have we recognized all the strains and species of Borrelia that cause Lyme disease symptoms, and 2) are we incorporating them into our tests? The answer is no. Convenience and expedience has chosen that we don't prime our ELISA tests withwild strains, but use a laboratory strain.
W hen a lab reports that their ELISA test has had high specificity and high sensitivity, it is usually interpreted by doctors as being a more accurate test, but the doctors don't know what the lab is actually measuring. One of the hidden problems of serologic Lyme tests is the fact that the tests must be primed with a source of bacteria to create the reactions with the patient's antibodies. To do this, virtually all labs rely on a laboratory strain of Bb known as strain B-31.Taking purified antigens from strain B-31 and injecting them into mice, they then can extract a monoclonal antibody to each antigen, or a polyvalent antibody soup. This antibody is concentrated and purified, and then added to the ELISA test to test the efficacy and performance of the test. Unlike the wild strains, B-31 grows well in culture, and this makes it a perfect choice as a consistent and inexpensive source of Bb. But the affinity the mouse monoclonal antibody has to B-31 antigen is quite different from the affinity the patients' antibodies have to the same antigen. This means the test may register as negative because the test cannot detect the slightly different antibody profile that a wild strain of Bb can produce. In other words, the labs are really comparing apples to oranges! This is why, when the American College of Pathologists used human sera to test the accuracy of 516 different laboratories ELISA tests nation wide, the overall accuracy was only 45%.
In the quest for specificity, most ELISA tests have become so specific that the test may fail to detect antibodies from related strains of Borrelia. This would include different genospecies that cause Lyme disease, as well as different Borrelia species that cause Tickborne Relapsing Fever. Would a cross reaction to the Borrelia species that cause Tick-borne Relapsing Fever be so bad?
The real Achilles' Heal of an ELISA Test is that it can only detect free antibody. It cannot detect any antibody that has become complexed with antigen.
The ELISA test depends on the active, free antibodies to attach to the free antigens that have been embedded on the walls of the test tube. If the antibodies in the serum being tested are already attached to antigens, then the enzyme reaction cannot take place. If we think of antibodies as sort of keys that fit into locks, and that on the surface of the bacteria are specific locks we now call antigens, you can see that once a key is inserted into a lock, the key is no longer available to open any other locks.
What makes this test so misleading is that many doctors accept high readings as an indication that the patient must really be sick. This logic is exactly backwards. If a patient is really infected with lots of bacteria, that means they have a lot of bacterial antigens floating around in the blood that are complexing free antibodies. So, as free antigen increases, free antibody decreases. Since the ELISA test detects only free antibody, a negative test might actually indicate a more serious infection. Many times, I have seen totally asymptotic patients with ELISA titers over 1000 be treated as though they were on death's doorstep simply because they had a high titer, while patients with borderline titers who are practically disabled are ignored, because a low titer is perceived as meaning less infected! These conclusions are erroneous and actually opposite to the truth, which is that a high titer means greater natural immunity.
This phenomena can actually be observed by using vaccines. If a patient has been vaccinated for a disease like tetanus, they will carry a high titer of free antibodies. If you try to measure those antibodies an hour after a booster shot is given, they will test negative. This is because the injected tetanus antigen complexes all available free antibody before the body can make more, so the measurable free antibody level drops.
The nature of all antibody is to seek out the proper antigen. The level of free antibody available is variable and often inadequate for the amount of antigen available. As antigen increases (i.e. The bacteria are dividing faster than the immune system can handle), free antibody drops.
What a high ELISA test may be a better indicator of is what level of immunity is the patient capable of mounting against this infection? A high titer is the same thing as saying the patient has a high natural immunity, and a low can mean that the patient may be overwhelmed by infection.
In one year-long study by Dr. Sam Donta, MD, done on chronic Lyme patients, the initial ELISA tests proved to be more than 66+% inaccurate (1996 LDF Conference lecture). Other researchers have also found the ELISA tests to be inaccurate. Using a 45- panel diagnostic testing protocol from the NIH for testing the efficacy of the ELISA and Western Blot, researchers found the accuracy of the Lyme ELISA varied from about 5075%, and were routinely inconsistent. The CDC's ELISA test did no better on average than any other ELISA. It is the CDC ELISA test which is used for surveillance of emerging Lyme disease in the United States, yet the test was correct only about two out every three tests. Too often, a single negative ELISA test can prevent a sick patient from getting treatment, even despite having serious symptoms!
In my opinion, the ELISA test is worthless as a diagnostic tool in Lyme disease. It is inconsistent and inaccurate, and should be discontinued as a tool to diagnose Lyme. If the NIH and CDC truly believe, as they've stated, that the diagnosis of Lyme disease is to be made on the basis of symptoms, then these tests should be temporarily banned until each manufacturer can prove efficacy using human serum.
-------------------- There is no wealth but life. -John Ruskin
All truth goes through 3 stages: first it is ridiculed: then it is violently opposed: finally it is accepted as self evident. - Schopenhauer Posts: 5639 | From Aptos CA USA | Registered: Apr 2005
| IP: Logged |
treepatrol
Honored Contributor (10K+ posts)
Member # 4117
posted
IGG by Western Blot P41 Present All other bands say absent Lyme IgG WB Interpreted. NEGATIVE The Igg is negative.
IgM by Western Blot P41 Present P39 Present Lyme IgM WB Interpreted. POSSITIVE IgM is positive for two bands 41 = Flagella or tail. This is how Borrelia burgdorferi moves around, by moving the flagella. Many bacteria have flagella. This is the most common borreliosis antibody.
and 39 is a major protein of Bb flagellin; specific for Bb p39 is a protein highly specific of the Borrelia genus.
And most of all it should be diagnosed clinically! relating to, based on, or characterized by observable and diagnosable symptoms of disease
-------------------- Do unto others as you would have them do unto you. Remember Iam not a Doctor Just someone struggling like you with Tick Borne Diseases.
treepatrol
Honored Contributor (10K+ posts)
Member # 4117
posted
93 kDa p100: chromosomal protein highly specific of the Borrelia genus; the antibody response is mostly IgG and appears in the course of chronic infections; bands at 93 kDa are strongly associated with advanced stages. 73 kDa Specific marker, probably corresponding to the bacterial DNA. 62-72 kDa Heat shock proteins; these bands are not specifically associated with a Borrelia infection and are found in several bacterial infections. No diagnostic value. 66 kDa Species-dependant but specific marker for Borrelia infections. 60 kDa Common bacterial antigen; non-specific. No diagnostic value. 58 kDa Species-dependant but specific marker for Borrelia infections. 46 kDa Species-dependant but specific marker for Borrelia infections. 41 kDa Flagellin: this protein is not specific to the Borrelia genus but is useful in the test interpretation; cross-reaction with other spirochetes are common; a flagellin positive reaction may occur at early as well at late stages. 39 kDa p39 is a protein highly specific of the Borrelia genus. 37 kDa This protein is considered to be an early marker but its specificity has not been established. 32.5 kDa OspA: surface protein highly specific of Borrelia garinii. 28 kDa OspD: surface protein. 22-23 kDa OspC: surface protein highly specific of Borrelia garinii and a marker for early infections. 18 kDa p18: the specificity of this protein has been recently established; excellent marker for late infections. From NovaTech
IgG WESTERN BLOT
The IgG Western Blot is a sandwich-type immunoassay performed in a manner that allows visualization of the patient's antibodies. It is a qualitative test and is generally more sensitive and specific than the ELISA. This test must be used if the Lyme IgG/IgM antibody serology is equivocal or positive. The somewhat-specific Lyme antibodies of importance are against the following molecular weights of the B. burgdorferi antigens: 23-25 kDa (Osp C); 31 kDa (Osp A); 34 kDa (Osp B); 39 kDa; 41 kDa; and 83-93 kDa7. "kDa" is the abbreviation for "kilodalton," which is used for molecular weight designations. "Osp" refers to outer surface protein of the bacteria.
There are currently multiple criteria that support a positive blot. "Positive" means that certain antibodies to B. burgdorferi are present. The CDC/ASTPHLD criteria are very conservative, require 5 of 10 bands (antibodies) for a positive result, and do not recognize equivocal or borderline results.8,9 These criteria would be more appropriate for a formal clinical study during early Lyme disease.
IGeneX has several years of clinical data that support more liberal reporting criteria.10 In addition, current studies show that the CDC/ASTPHLD criteria miss some patients with culture-proven erythema migrans (EM).5,11 Both the IGeneX and the CDC/ASTPHLD criteria are included on the IGeneX report form sent to the physician. 3,5,8,9
The Western Blot involves a highly complex visual determination of protein bands, based on their molecular weights and intensities. The IGeneX report form provides an interpretation along with the results in detail.
A positive IgG result with clinical history may be indicative of Lyme disease. Patients with other spirochetal disease and/or who test positive for rheumatoid factor or Epstein Barr virus may have cross-reacting antibodies. A positive response in this, as in any antibody assay, indicates sensitization, not necessarily active disease.
IgM WESTERN BLOT
The IgM Western Blot is a very sensitive indicator of exposure to B. burgdorferi. It may be positive as early as one week after a tick bite, and will usually remain positive for six to eight weeks after the initial exposure. Re-exposure and recurrent disease also cause this test to be positive for a period of time. For the testing to be complete, the IgM blot should be run along with the IgG blot. However, for economic reasons, the IgG blot may be run first: when the IgG blot is negative, the IgM blot should be performed.
The antibody specificities of importance for the IgM blot are similar to those for the IgG blot (with the exception of 83-93 kDa, which is still being investigated for significance). The CDC/ASTPHLD criteria for a positive result are two of the following three bands: 23-25 kDa (Osp C); 39 kDa; and/or 41 kDa.8,9 IGeneX adds the 31 kDa (Osp A), and/or the 34 kDa (Osp B) to the criteria,10,12 with the argument that these two antigens are used for the vaccines and therefore their antibodies should be included in the interpretation of positivity. The IgM Western blot is often positive in patients with persistent infection.6 Sometimes it is the only antibody marker detected.
When the IgM ELISA is equivocal or positive, the IgM Western blot must be performed. In addition, because the literature suggests that rheumatoid conditions may lead to false positive IgM antibody responses, an ANA/DNA/rheumatoid factor screen may be ordered to rule out false positive reactions. Patients testing positive with serologic tests for syphilis may also test positive for the Lyme antibody tests.13-15
A positive IgM result with clinical history may be indicative of early Lyme disease or persistent infection in otherwise serologically negative individuals. Recently reported data support our observation that some Lyme patients may have only a restricted IgM response to B. burgdorferi. 16,17
Similar to the IgG Western blot, the IgM Western blot involves a highly complex visual determination of protein bands, based on their molecular weights and intensities. For both tests, IGeneX uses multiple negative controls to serve as baselines for comparison to positive responses.
-------------------- Do unto others as you would have them do unto you. Remember Iam not a Doctor Just someone struggling like you with Tick Borne Diseases.
Michelle M
Frequent Contributor (1K+ posts)
Member # 7200
posted
Hi again, FedUp.
I reviewed some of your other messages to try and get more info and insight. You have said:
quote:Does anyone know why a western blot lyme test would show a false positive result even though all the symptoms of lyme are present?????
My daughter's dr. said she tested positive for lyme (she has been very sick for a year now), but it may be a false positive result. Any suggestions would be appreciated
And so forth...
Aha! The old "false positive" duck opinion! I knew it!
You have really got to get your daughter to an LLMD (lyme literate medical doctor).
Ducks (our name for doctors who refuse to educate themselves about lyme) are NEVER going to help your daughter.
You literally have to become a lyme advocate yourself. Read, read read and then read some more.
Learn more than the Duck knows! Fortunately, this is not hard.
He believes her test is "false positive" because he is relying on the tired and useless negative ELISA. Well, guess what? The ELISA misses over half of all proven cases of chronic lyme. Does your duck know this? Unlikely.
Has your Duck even mentioned co-infection testing? Nope. Didn't think so. Do you know what babesia or bartonella can do to a person, untreated? Estimates are that as high as 60% of people with lyme have at least one co-infection.
If you want your daughter to get better -- you have GOT to get away from this doctor and find one who knows what he's doing.
Yes, there is a lot of reading and learning involved. But you must get started.
Not trying to be harsh; however, almost all of your posts ask the same questions. You've been given GREAT, science-backed answers, but you don't seem to get back on the threads to acknowledge them!
Sometimes new posters don't know what happened to their posts when they slip off the front page. Is it possible you've lost your other postings and haven't seen the responses?
Please get your daughter to an LLMD. Her health and future are at stake and it is worth whatever it takes.
Michelle
Posts: 3193 | From Northern California | Registered: Apr 2005
| IP: Logged |
The Lyme Disease Network is a non-profit organization funded by individual donations. If you would like to support the Network and the LymeNet system of Web services, please send your donations to:
The
Lyme Disease Network of New Jersey 907 Pebble Creek Court,
Pennington,
NJ08534USA http://www.lymenet.org/
UBBFriend: Email this page to someone!
Printer-friendly view of this topic