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.