≡ Menu

ELISA Test for Lyme Disease

Lyme Disease TestThe most common Lyme disease test is the enzyme-linked immunosorbent assay (ELISA).  This test has been available for Borrelia burgdorferi since 1984 and most commercial ELISA tests use a whole cell sonicate of the bacteria.  The ELISA test detects antibodies to the Borrelia burgdorferi bacteria that cause Lyme disease and results are usually reported in titers indicating the level of antibodies in the sample.  The ELISA test is primed, using laboratory strains of Borrelia burgdorferi, by breaking down the antigens into fragments and embedding them in the side of the reagent vessel (such as a test tube).  The patient’s serum (blood) or cerebrospinal fluid (CSF) is added and any antibodies contained in the sample which are specific to the test strain will bind to the antigens.  This bond is indicated by special enzymes linked to the antigens which change color in the event of an antibody-antigen reaction.  A high titer, of say 1:256, means that the sample requires considerable dilution, in this case one part serum to 256 parts water, before the reaction no longer occurs and no color change is detectable.

Sensitivity of ELISA Testing

The ELISA test is considered to have the highest sensitivity in detecting Lyme disease but a fairly low specificity, which is the reason that guidelines from bodies such as the CDC advise follow-up testing with a Western blot should the result of the ELISA test be either positive or indeterminate.  Where an ELISA test is unavailable, an immunofluorescence assay can be performed but this is considered less reliable than the ELISA test by the CDC.

The original ELISA test involved in two-tier testing determines the serum antibody levels of immunoglobulin M and immunoglobulin G.  These antibodies arise in response to infection with the IgM usually reaching detectable levels within two to four weeks and the IgG antibodies able to be detected between four and six weeks with a peak at eight weeks.  The timing of ELISA tests for IgG and IgM is, therefore, very important in order to ensure that a relatively early-stage infection or late stage infection, where IgM levels may have dissipated, is not the cause of a negative result. 

Advances in ELISA Lyme Disease Testing

A more recent ELISA test for Lyme disease take the form of the C6-peptide ELISA which detects IgG antibodies to the variable major protein-like sequence-expressed (VIsE) sixth invariate region (C6) peptide of the Borrelia bacteria.   This test appears to be more sensitive and specific than previous ELISA tests particularly in cerebrospinal fluid samples.  New generation ELISA tests aim to be more sensitive to the unique and specific Borrelia burgdorferi antigens such as OspA (31kDa), OspB (34kDa), OspC (23025kDa), 39kDa, and 93kDa.

Problems with the ELISA Test for Lyme Disease

In addition to the necessary timing considerations for the ELISA tests, it is also important that the test is primed with the appropriate strains of Borrelia bacteria for each case.  With the standardization of most commercial testing this is rarely done meaning that false-negatives are much more likely to occur than with patient-tailored testing.  Borrelia bacteria are highly heterogeneous with significant differences in surface proteins occurring during cell division amongst the bacteria.  This is a survival technique employed by the bacteria to avoid detection by our immune systems and, as such, the antibodies produced in response to one strain of Borrelia burgdorferi sensu lato may not correspond to the outer surface proteins of a different strain. 

Lyme Disease in Europe – Testing Times

The differences in OSPs found on Lyme disease bacteria poses problems for patients in Europe where several strains of Borrelia bacteria are known to cause Lyme disease, including B. afzelii, and B. garinii amongst others.  All of these strains require inclusion in ELISA testing in Europe making a single test against a single antigen highly questionable.  In the US and Canada a similar problem arises in that the laboratory strains used in the ELISA test may have significant polymorphic differences to the wild-strains of Borrelia burgdorferi, thus giving a false-negative in the presence of Lyme disease.  Further research on the strains present in the current environment is required to ensure that all Borrelia bacteria capable of inducing Lyme disease are identified and are thus able to be incorporated into antigen testing.

Problems with Animal Testing

The majority of Lyme disease test centers in US laboratories rely on a strain of Borrelia bacteria known as B-31.  This strain is injected into mice in the laboratory in order to then extract a monoclonal antibody to each antigen which can be used to determine the accuracy of the ELISA test.  A further potential problem may be posed by the slightly different responses of the immune system of mice and humans, making it difficult to assess the accuracy of the test method.  A mouse monoclonal antibody does have a different affinity to the B-31 strain, and presumably other Borrelia strains, than a human serum antibody which is why the American College of Pathologists carried out a study to determine the accuracy of ELISA tests using human sera.  The results of their cross-laboratory study found an overall accuracy of just 45% for the 516 testing facilities investigated (Bakken, et al, 1997).

Is a Low Lyme Titer Actually Proof of the Immune System’s Hard Work?

Even assuming that no problems exist in using animals in Lyme disease research such as this, the increased specificity of the ELISA test can mean that a test fails to identify antibodies to strains similar to that being tested but not exactly the same.  The ELISA test is also only able to detect free antibodies, meaning that those present in the sample that have already complexed with an antigen present in the patient will not show up in testing, thus leading to a false-negative.  Somewhat counter-intuitively, those patients with high titers in an ELISA test are often considered to have a severe infection whereas those with low levels of antibodies may be classed as disease-free.  In the latter case however, it may be that high levels of antigen have been successfully complexed by antibodies leaving few free antibodies to react with a test sample.  Although the patient’s own antibodies may then successfully eradicate the infection this does not mean that their infection is not serious but it may, due to a negative ELISA test, simply be ignored.  A high titer may actually indicate a stronger natural immunity rather than extreme infection.

Continue Reading –> ELISA Accuracy