Saturday, 3 September 2011


Large differences between test strategies for the detection of anti-Borrelia antibodies are revealed by comparing eight ELISAs and five immunoblots.


VUMC, Amsterdam, The Netherlands.


We investigated the influence of assay choice on the results in a two-tier testing algorithm for the detection of anti-Borrelia antibodies. Eighty-nine serum samples from clinically well-defined patients were tested in eight different enzyme-linked immunosorbent assay (ELISA) systems based on whole-cell antigens, whole-cell antigens supplemented with VlsE and assays using exclusively recombinant proteins. A subset of samples was tested in five immunoblots: one whole-cell blot, one whole-cell blot supplemented with VlsE and three recombinant blots. The number of IgM- and/or IgG-positive ELISA results in the group of patients suspected of Borrelia infection ranged from 34 to 59%. The percentage of positives in cross-reactivity controls ranged from 0 to 38%. Comparison of immunoblots yielded large differences in inter-test agreement and showed, at best, a moderate agreement between tests. Remarkably, some immunoblots gave positive results in samples that had been tested negative by all eight ELISAs. The percentage of positive blots following a positive ELISA result depended heavily on the choice of ELISA-immunoblot combination. We conclude that the assays used to detect anti-Borrelia antibodies have widely divergent sensitivity and specificity. The choice of ELISA-immunoblot combination severely influences the number of positive results, making the exchange of test results between laboratories with different methodologies hazardous.

Serologic Tests for Lyme Disease Yield Disparate Results

When serum samples were tested for anti-Borreliaantibodies using eight commercially available ELISAs and five immunoblot assays, intertest agreement was only modest.

Current guidelines for diagnosing Lyme disease include a two-tier testing algorithm: an enzyme-linked immunosorbent assay (ELISA) for detecting anti-Borrelia antibodies, followed by immunoblot confirmation of positive ELISA results. Commercially available tests are based on sonicated whole-cellBorrelia antigens, recombinant antigens, or a mixture of the two.

To compare the performance of these assays, researchers in the Netherlands tested serum samples for anti-Borrelia antibodies using eight commercial ELISAs and five immunoblots (4 commercial, 1 investigator-made). The 89 samples were from 59 patients with suspected Lyme disease, 14 healthy controls, and 16 patients with syphilis or Mycoplasma pneumoniae infection — conditions associated with highly cross-reactive antibodies.

Of the 89 samples, 35 (39%) tested negative — and 16 (18%) tested positive — on all ELISAs. The remaining 38 (43%) tested positive on one to seven ELISAs. The proportion of samples with positive results on any one ELISA ranged from 34% to 59% for patients with suspected Lyme disease and from 0% to 38% for patients with cross-reactive antibodies. Samples from healthy controls almost always had negative ELISA results.

Thirty-one of the samples from patients with suspected Lyme disease were also tested with all immunoblots. Interblot agreement was low (IgG, 30%–84%; IgM, 0%–46%), and it was no higher for recombinant antigens than for whole-cell antigens. Some samples that tested negative on all ELISAs showed blot reactivity; some that tested positive on all ELISAs tested negative on all blots.

Comment: These findings are sobering and, unfortunately, do not facilitate diagnosis of Lyme disease. Clinicians should rely on a precise clinical determination of Lyme disease, interpreting serologic test results with great caution. Clearly, several possible ELISA/blot combinations do not work together very well. Furthermore, there are true Lyme cases with positive ELISA but negative blot results (depending on the test used), and even a few with negative ELISA but positive blot results.

Thomas Gl├╝ck, MD

link here


  1. This is indeed sobering. I wish some independent researcher would conduct the same examination of the US' own immunoblots and make a comparison between them and ELISA.

  2. I did read other research where different labs were looked at, I thought in US but can't remember where I read it may have been during IDSA review hearing