Western Blot Testing

The Western Blot Blood Test is the only way currently approved by the CDC to test for Lyme disease in the human body. The test was created many years ago, when Lyme was first discovered and being studied. Below I will discuss the basic scientific underpinnings of a Wester Blot test, some of the problems with the Lyme-specific Western Blot, and some explanations for both false positives and false negatives. The Western Blot is a rather complicated scientific process, and I have done my best to explain it in a way that makes sense. If you don’t want to read the more complicated explanations, the more in-depth sections have an italicized section at the button that acts as a summary.  

What is a Western Blot?

The Western Blot is a specific kind of biological assay, or test, that counts the amount of antibodies in your blood. Antibodies are proteins produced by your immune system to find and fight against various bacteria, viruses, and other lifeforms. Antibodies are highly specific – they are produced by your body in reaction to one unique protein.  Some proteins are common, shared by many bacteria or viruses, while some are only produced by a single organisms. Therefore some antibodies bind to more than one bacteria or virus, while others are highly specific and only bind to proteins from one species. The Western Blot Test evaluates antibodies for proteins found in Borellia Burgdorfi – some of these proteins are common to all bacteria, and some are only found in this specific species of bacteria. If you have these antibodies in your blood, your immune system has ‘seen’ Burgdorfi before, and has produced these antibodies to help protect you from it.

How Does it Work?

A Borellia Burgdorfi Lyme bacteria is cut into bits of protein, and those proteins are exposed to your blood to see if your immune system has made antibodies that match them. A match indicates that your immune system has ‘seen’ that protein before. 

Understanding the Results

The more Borrelia burgdorferi proteins your immune system has produced antibodies for, the more positive your test is considered to be. A nuance is that some labs add more weight to proteins that are Lyme-specific.  Of course, this test relies on the effectiveness of your immune system in forming antibodies. Not all bodies do this equally well. If your immune system doesn’t properly produce antibodies, you will not test positive even if you have been exposed to Lyme. Additionally, the test was first developed to guarantee that people in the first clinical trials studying Lyme treatments actually had Lyme. Thus, the criteria for a conclusive positive test is very high. GIven all this, you can imagine the potential for false negatives, or test results that say there is no conclusive Lyme presence, even in a person with strong evidence of infection.

Summary: The test is sometimes negative even in people who have Lyme disease.

Reasons for false negatives:

  • Lyme has spread through tick, human, and woodland vectors since its discovery. Along the way, Lyme has mutated and changed, so that Borellia Burgdorfi found in Virginia has proteins that are different from the proteins of Borellia Burgdorfi from Pennsylvania. Both bacteria are Lyme, but exposure to them will result in very different conclusions on a Western Blot Test, as the test is highly specific for the ‘original’ Lyme bacteria.
  • Testing is done too soon after the tick bite. It can take 4-­6 weeks for a detectable level of antibodies to develop.
  • The ill person has a poorly functioning immune system that isn’t producing detectable levels of antibodies.
  • The patient is or has recently been on steroids or certain anti­cancer drugs, which suppress the immune system.
  • The patient is or has recently been on antibiotics, which can reduce the production of antibodies.
  • The antibodies may already be bound to Lyme bacteria in your blood, with not enough free­-floating antibodies to respond to the proteins in the Western Blot Test.
  • The bacteria have changed their antigenic make­up, evading recognition by the immune system.
  • The patient has a variant strain of the disease that the test fails to detect.
  • There is no test that can prove a person doesn’t have Lyme disease.

Important Note: Because the Western Blot Test can be negative in people who would benefit from being treated for Lyme disease, the CDC does not recommend nor do I practice withholding antibiotics from people with a tick bite and symptoms, I believe that treatment should begin as soon as Lyme disease is suspected. Lyme is far less complicated to eradicate when treatment is started early.

 False Positives can also occur:

  • A person who has had an exposure to Lyme and has been treated adequately may test positive even though the person does not have any remaining Lyme.  If no new symptoms develop over the next few months they are likely fine.
  • Some people when exposed to Lyme form a strong enough antibody response to eradicate the Lyme without antibiotics.  We do not know how common this is.

Additional, in-depth information on the Western Blot:

Below are the proteins assessed in a Western Blot, and their function in a Lyme bacteria. For each of these proteins, the immune system produces IgM antibodies first, and IgG antibodies second. The current CDC criteria for a positive Western Blot are as follows:

Two of the following three bands have to be positive for IgM antibodies: OspC  23, 39 and 41.

Five of the following ten bands have to be positive for IgG antibodies: 18, OspC 23, 28, 30, 39, 41, 45, 58, 66 and 93.

 18 – Segment of flagellin, a protein used for movement by many different bacteria.

20 – Cross-reactive for Borellia Burgdorfi

21 – Unknown

22 – Specific for Borellia Burgdorfi

23-25 – OspC (outer surface protein C), specific for Borellia Burgdorfi

28 – OspD (outer surface protein D), specific for Borellia Burgdorfi

30 – Segment of OspA (outer surface protein A), common in both European and California strains of Borellia

31 – Segment of OspA (outer surface protein A), specific for Borellia Burgdorfi, this protein is used for vaccines so is not included in most testing

34 – OspB (outer surface protein B), specific for Borellia Burgdorfi, this protein is used for vaccines so is not included in most testing

35 – Specific for Borellia Burgdorfi

37 – Specific for Borellia Burgdorfi

38 – Cross-reactive for Borellia Burgdorfi

39 – Segment of Borellia Burgdorfi flagellin specific for Borellia Burgdorfi

41 – Segment of flagellin, this protein occurs in all spirochetes and is often the first to appear after a Borellia Burgdorfi infection

45 – Cross-reactive for all Borellia (sometimes people with Lyme who have this band positive also have the co-infection Ehrlichiosis)

50 – Cross-reactive for all Borellia

55 – Cross-reactive for all Borellia

57 – Cross-reactive for all Borellia

58 – Unknown but may be a heat-shock Borellia Burgdorfi protein

60 – Cross reactive for all Borellia

66 – Cross-reactive for all Borellia, common in all bacteria

83 – Specific antigen for the Lyme bacterium, probably a cytoplasmic membrane

93 – Unknown (protentilly the same protein as in band 83, which may migrate differently in some patients)

 

How A Western Blot Test is Performed:

First Stage

  • Borellia Burgdorfi bacteria are taken from a lab and these bacteria are treated so that the proteins on the surface of their cells, the ones your immune system responds to, are separated from the rest of the cell matter and from each other so that they are floating independently in a solution,
  • Your blood sample is added to this mixture, containing any antibodies produced by your immune system,
  • The antibodies in your blood bind to any available ‘matching’ protein 

Second Stage

  • Once your antibodies have bound to the corresponding Lyme proteins, they need to be tagged so that they can be analyzed,
  • All antibodies share the same shape on the end that does not capture bacteria, and this shape changes once they have bound to a protein,
  • Lab-manufactured antibodies are added to the mixture. Rather than binding to proteins in bacteria, viruses, etc, these antibodies are constructed by biologists to bind to antibodies in the shape shared by all antibodies that have bound to a protein in the previous stage,
  • Once tagged in this way, the lab made antibodies that have ‘captured’ your antibodies are separated from the rest of the mixture,

Third Stage

  • The protein-antibody-antibody triplets are put on a special plate and stimulated with electric current,
  • This current pushes proteins down the plate, but affects proteins of different sizes in different ways, so that each protein is separated from the others based on its size,
  • The sizes of the Lyme proteins involved in this test are known, and marked on the plate,
  • If there are proteins in a band on the plate in an area that corresponds with a Lyme protein, then you have tested positive for antibodies that bind to that Lyme protein