Outer surface protein C peptide derived from Borrelia burgdorferi sensu stricto as a target for serodiagnosis of early lyme disease

Abstract

Current serodiagnostic assays for Lyme disease are inadequate at detecting early infection due to poor sensitivity and nonspecificity that arise from the use of whole bacteria or bacterial proteins as assay targets; both targets contain epitopes that are cross-reactive with epitopes found in antigens of other bacterial species. Tests utilizing peptides that contain individual epitopes highly specific for Borrelia burgdorferi as diagnostic targets are an attractive alternative to current assays. Using an overlapping peptide library, we mapped linear epitopes in OspC, a critical virulence factor of B. burgdorferi required for mammalian infection, and confirmed the results by enzyme-linked immunosorbent assay (ELISA). We identified a highly conserved 20-amino-acid peptide epitope, OspC1. Via ELISA, OspC1 detected specific IgM and/or IgG in 60 of 98 serum samples (62.1%) obtained from patients with erythema migrans (early Lyme disease) at the time of their initial presentation. By comparison, the commercially available OspC peptide PepC10 detected antibody in only 48 of 98 serum samples (49.0%). In addition, OspC1 generated fewer false-positive results among negative healthy and diseased (rheumatoid arthritis and positive Rapid Plasma Reagin [RPR+] test result) control populations than did PepC10. Both highly specific and more sensitive than currently available OspC peptides, OspC1 could have value as a component of a multipeptide Lyme disease serological assay with significantly improved capabilities for the diagnosis of early infection.

Fig 1

IgG and IgM antibodies specific for OspC peptides in sera from patients with Lyme disease. Sera from patients with Lyme disease were confirmed to be positive for anti-Borrelia antibodies using commercially available Western blot strips prior to incubation with OspC peptides (10 μg/ml) in an ELISA. Antibody binding was detected using a polyclonal HRP-labeled goat anti-human IgG and IgM (γ- and μ-chain-specific) antibody. Upper panels show dose titration of Lyme disease patient sera (Lyme sera, n = 10) and healthy control sera (normal sera, n = 10) on OspC peptide-coated 96-well plates. Data are reported as mean absorbance ± SD. Lower panels depict binding of serum from Lyme disease patients and healthy controls (normal sera) at a single dilution of 1:100. Data are reported as absorbance at 450 nm; the solid lines represent means ± SD. Numbers of samples: for OspC1, Lyme disease, n = 20; normal, n = 10; for OspC 18, Lyme disease, n = 20; normal, n = 10; for OspC30, Lyme disease, n = 17; normal, n = 10. Patient samples whose results are depicted in the upper panels are different from those used in the lower panels. *, P < 0.05 by the Mann-Whitney test.

Fig 2

Amino acid sequence alignment of different OspC types depicting the regions corresponding to OspC1, OspC30, and PepC10. Sequences were aligned using CLC Workbench and were trimmed to show only the regions corresponding to the peptides of interest. In several instances, complete sequences for the OspC types containing all three peptides were not available. When possible, multiple partial sequences for that OspC type were aligned, depicting the presence or absence of a particular peptide sequence. *, partial sequence; **, partial sequence used for epitope mapping. B. pacificus, B. burgdorferi strain isolated from Ixodes pacificus.

Fig 3

Comparison of OspC1 and PepC10 in the detection of IgM and IgG antibodies in sera from patients with early Lyme disease. Sera from patients with early Lyme disease (erythema migrans positive [Lyme]), healthy controls (normal), patients with rheumatoid arthritis (RA), RPR+ patients, or patients with Lyme arthritis (late LD) were incubated on OspC1- or PepC10-coated (10 μg/ml) plates in an ELISA. Serum IgM (a) and IgG (b) was detected using HRP-labeled goat anti-μ or anti-γ chain antibodies, respectively. The dashed line represents 3 SD from the mean of healthy controls incubated with OspC1; the dotted line represents 3 SD from the mean of healthy controls incubated with PepC10. The lines overlap in panel a. The y axis in panel b is segmented to show the data more clearly. Data are reported as absorbance at 450 nm. Numbers of samples: Lyme disease, n = 98; normal, n = 48; RA, n = 48; RPR+, n = 39; late Lyme disease, n = 20.