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Lyme Disease Test Accuracy – Western Blot

Western Blot Test

Sample of Western Blot Test Results

The Western blot uses a fairly simple technique which sorts antibodies by the relative weight of their antigens, thus separating them out for analysis. The units used to differentiate these ‘bands’ are kilo daltons (kDa), a tiny unit of weight approximately equal to the mass of one proton or one neutron. Gel electrophoresis is used to separate native or denatured proteins according to the length of the polypeptide or the structure of the protein in the sample. These proteins are then transferred to a membrane where antibodies specific to the target protein are used to detect them.

Results of a Western blot test for Lyme disease are separated into bands such as 22kDa, 41kDa, 93kDa, and so on, with some representing significant markers of Lyme disease antibodies. The 31kDa band on the Western blot indicates the presence of an antibody to OspA and is specific to just a few strains of Borrelia bacteria (including that used in the LYMErix vaccine). Other significant bands are 34kDa (OspB) and 23kDa (OspC).

Lyme Disease Test Bands Revised Amidst Confusion

The potential for confusion and inconsistency in reporting and analysis of the Western blot test, given the multitude of bands involved, the CDC and the Association of State and Territorial Public Health Laboratory Directors revised guidelines in 1994. The new guidelines no longer considered bands 22, 23, 25, 31, and 34kDa to be specific and significant for Borrelia burgdorferi bacteria exposure and removed these bands from even being reportable, much to the chagrin of a number of Lyme disease researchers and advocates at the time. No reason was given for this action and a previously reasonable test for detecting Lyme disease was now considered fairly poor by many clinicians. A number of laboratories also stopped reporting which bands were highlighted and began merely to give a positive or negative interpretation along with the new guidelines.

Research carried out by Fawcett, et al (1995), highlighted the problem with the new guidelines by using confirmed cases of Lyme disease to evaluate the accuracy of the old Western blot and the updated version. The new guidelines included ten specific bands as reportable out of the original twenty-five. For an IgG Western blot a positive result was determined as at least five of the following bands: 18, 21,28, 30, 39, 41,,45, 58, 66 and 93kDa. Two or more of the bands 23, 39, and 41, were needed for a positive IgM Western blot.

New Bands Not Specific Enough for Lyme Test

The researchers investigating the new guidelines were troubled that several of the bands previously considered specific and significant were no longer present, namely 22, 23, 25, 31, and 34, which included OspA, OspB, and OspC. Sixty-six symptomatic paediatric patients with a history of tick bite, and erythema migrans tested positive under the old guidelines whilst just twenty were deemed positive on the new criteria. This potential level of inaccuracy would then lead to significant underdiagnosis and denial of treatment for Lyme disease for many patients.

The lack of change in the CDC guidelines since 1995 is surprising considering the amount of research carried out into Lyme disease and its interaction with the immune system. Whilst the guidelines are still considered incomplete by some so-called ‘Lyme literate medical doctors’ (LLMDs) no further suggestions of substantial revision have been accepted by the CDC. Support for the current two-tier testing method has been criticised however, with one paper by Steere, et al (2008), claiming 100% sensitivity despite a positive test being part of the criteria for a diagnosis (i.e. circular reasoning was being employed, whether disingenuously or by oversight). Steere himself suggests, amongst others, that an alternative C6 peptide ELISA test merits more investigation as a possible replacement of the current two-tier types of Lyme disease tests.

European Western Blot Test Accuracy

The current guidance concerning the use of Western blot testing for Lyme disease in Europe is formulated by European Concerted Action on Lyme Borreliosis using interpretation criteria from Engstrom, et al (1995) and Dressler, et al (1993), for IgM and IgG respectively. The reason that the criteria differ from the US is that different strains of Borrelia bacteria are present in Europe. The EUCALB guidance also states that the use of these recommended criteria is open to interpretation depending on the strain or species used as an antigen. Eight bands were identified as discriminatory for Lyme Borreliosis detection although these varied in significance.

Engstrom’s guidance suggests a positive IgM Western blot when two out of three proteins (24, 39, and 41kDa) are recognized in the sample. Dressler recommends discrimination on the basis of at least five out of the ten most common IgG bands in early Lyme disease: 18, 21, 28, 30, 39, 41, 45, 58, 66, and 93kDa. This IgG blot was shown to have a specificity of 95% and a sensitivity of 83% after the first weeks of infection. The guidance on interpreting and reporting Western blot results in Europe has not changed since the early to mid 1990s, just as the North American guidelines published by the CDC have not changed in recent years.

Continue Reading –> ELISA Accuracy


Dressler F, Whalen JA, Reinhardt BN, Steere AC. Western blotting in the serodiagnosis of Lyme disease. J Infect Dis. 1993 Feb;167(2):392-400.

Engstrom SM, Shoop E, Johnson RC. Immunoblot interpretation criteria for serodiagnosis of early Lyme disease. J Clin Microbiol. 1995 Feb;33(2):419-27.

Fawcett, P., et al. Western Blot and False Negatives in Children: Rheumatology Symposia Abstract #1254, 1995.

Mavin S, McDonagh S, Evans R, Milner RM, Chatterton JM, Ho-Yen DO. Interpretation criteria in Western blot diagnosis of Lyme borreliosis. Br J Biomed Sci. 2011;68(1):5-10.

Steere AC, McHugh G, Damle N, Sikand VK. Prospective study of serologic tests for lyme disease. Clin Infect Dis. 2008 Jul 15;47(2):188-95.

Hauser U, Lehnert G, Wilske B. Validity of interpretation criteria for standardized Western blots (immunoblots) for serodiagnosis of Lyme borreliosis based on sera collected throughout Europe. J Clin Microbiol. 1999 Jul;37(7):2241-7.