A Limitation of 2-Stage Serological Testing for Lyme Disease: Enzyme Immunoassay and Immunoblot Assay Are Not Independent Tests

To improve the accuracy of testing for antibody to Borrelia burgdorferi, 2-stage conditional testing has been recommended, in which sera that yield positive or equivocal results in a first-stage test (e.g., an ELISA) are then tested by immunoblot assay. The increased specificity anticipated with sequential testing, however, depends on immunoblot assays and ELISAs being independent tests. To examine whether they are independent, control serum samples were tested with 2 different commercially available IgM ELISAs and with an IgM immunoblot assay kit. The frequency of false-positive IgM immunoblot assays was significantly higher with ELISA-reactive than with ELISA-negative serum samples (P</=.001). In addition, there was a highly significant direct correlation between the number of reactive bands on IgM blotting and the rate of false-positive results by IgM ELISA (P<.0001). These observations demonstrate that IgM ELISAs and IgM immunoblot assays for antibodies to B. burgdorferi are not independent tests. Therefore, when used in sequential testing for Lyme disease, the immunoblot assay should be considered a test that supplements rather than confirms an ELISA.