Epitope Length, Genospecies Dependency, and Serum Panel Effect in the IR6 Enzyme-Linked Immunosorbent Assay for Detection of Antibodies toBorrelia burgdorferi

Abstract

In the absence of erythema migrans, the basis for diagnosis of Lyme disease is the demonstration of an antibody response against Borrelia burgdorferi in an appropriate clinical setting. The C6 enzyme-linked immunosorbent assay, based on the IR6 region of VlsE, has become widely used in both the United States and Europe. We mapped the antigenic epitopes of IR6 to a shorter sequence that is equivalent in sensitivity and specificity to the full-length IR6 25-residue peptide. In addition, we observed significant differences in sensitivity between serum panels (60 to 100%), indicating that the selection of the serum panels can shape the apparent overall sensitivity of the assay. Contrary to prior reports, the assay sensitivity is greater when the IR6 peptide is derived from the sequence of the same infecting Borrelia genospecies. Using our North American panels and the two panels obtained from European Lyme disease patients, we determined that the IR6 assay that is based on a single genospecies of Borrelia spp. is not optimal for use as a universal diagnostic assay for Lyme disease.

Outer Surface Protein C Peptide Derived from Borrelia burgdorferi Sensu Stricto as a Target for Serodiagnosis of Early Lyme Disease

“…The use of a single epitope increases the likelihood of generating false negatives, as there is only a single antibody binding site and a relatively small variation in the amino acid sequence at that site would decrease the ability of such an assay to detect infection. C6, which is more variable than originally recognized, places the IR6 assay at risk for this limitation (19,23,24). Despite the recent results, it is unlikely that the current paradigm will be changed in favor of the single-peptide IR6 assay.…”

“…An ELISA (IR6) using a peptide derived from the VlsE protein of Borrelia has demonstrated greater specificity in the detection of Lyme disease than whole-cell ELISAs and has been approved for use by the FDA (19,23). However, C6, the peptide derived from VlsE, does not bind IgM particularly well, is derived from an antigen that is expressed only after infection is established (fewer than 1% of bacteria in the tick express VlsE, and transcription of the gene is suppressed prior to transmission of the bacteria), and the IR6 region of VlsE from which the peptide is derived has shown a greater degree of variability than originally thought (19,(23)(24)(25)(26). Though the IR6 assay represents a significant improvement, in terms of specificity, compared to the whole-cell ELISA (27), these concerns have precluded the use of the IR6 assay as a stand-alone diagnostic test for early Lyme disease.…”

Borrelia burgdorferi Sensu Lato Species in Europe Induce Diverse Immune Responses against C 6 Peptides in Infected Mice

“…The B. gariniiderived C 6 peptide did not detect antibodies against B. afzelii or B. burgdorferi sensu stricto, as well as the just-mentioned B. afzelii-and B. burgdorferi sensu stricto-derived C 6 peptide variants (41). Regarding European patients' sera, it was proposed that the use of a C 6 peptide mixture might be more beneficial with regard of genospecies dependency and that early infections may not be detected by all C 6 test preparations (12,49). Previously reported data showed differences in the amino acid sequences between IR6 within the B. burgdorferi sensu lato complex (13), which might result in different reactivities of patient sera.…”

Decorin Binding Proteins A and B in the Serodiagnosis of Lyme Disease in North America

“…One method for improving the specificity of first-tier assays, and therefore eliminating the need for less sensitive second-tier assays, is through the use of synthetic peptides that contain epitopes unique to prominently expressed B. burgdorferi antigens as assay targets (9,13,(13)(14)(15)(16)(17). ELISAs utilizing synthetic peptides (PepC10 and C6) have demonstrated improved sensitivity for the serological diagnosis of acute Lyme disease compared to that with conventional two-tier testing (7,13,15,18,19).…”

“…ELISAs utilizing synthetic peptides (PepC10 and C6) have demonstrated improved sensitivity for the serological diagnosis of acute Lyme disease compared to that with conventional two-tier testing (7,13,15,18,19). Improved specificity provided by synthetic peptides may be, in part, due to the elimination of conserved structural motifs and/or linear amino acid sequences found in similar proteins expressed by other bacterial species, although this has never been directly proven.…”