Rapid, Simple, Quantitative, and Highly Sensitive Antibody Detection for Lyme Disease

Abstract

There is currently a need for improved serological tests for the diagnosis and monitoring of Lyme disease, an infection caused by Borrelia burgdorferi. In the present study, we evaluated luciferase immunoprecipitation systems (LIPSs) for use for profiling of the antibody responses to a panel of B. burgdorferi proteins for the diagnosis of Lyme disease. Initially, serum samples from a cohort of patients and controls (n ‫؍‬ 46) were used for training and were profiled by the use of 15 different B. burgdorferi antigen constructs. For the patient sera, the antibody responses to several B. burgdorferi antigens, including VlsE, flagellin (FlaB), BmpA, DbpA, and DbpB, indicated that the antigens had high levels of immunoreactivity. However, the best diagnostic performance was achieved with a synthetic protein, designated VOVO, consisting of a repeated antigenic peptide sequence, VlsE-OspC-VlsE-OspC, Analysis of an independent set of serum samples (n ‫؍‬ 139) used for validation showed that the VOVO LIPS test had 98% sensitivity (95% confidence interval [CI], 93% to 100%; P < 0.0001) and 100% specificity (95% CI, 94% to 100%; P < 0.0001). Similarly, the C6 peptide enzyme-linked immunosorbent assay (ELISA) also had 98% sensitivity (95% CI, 93% to 100%; P < 0.0001) and 98% specificity (95% CI, 90% to 100%; P < 0.0001). Receiver operating characteristic analysis revealed that the rates of detection of Lyme disease by the LIPS test and the C6 ELISA were not statistically different. However, the VOVO LIPS test displayed a wide dynamic range of antibody detection spanning over 10,000-fold without the need for serum dilution. These results suggest that screening by the LIPS test with VOVO and other B. burgdorferi antigens offers an efficient quantitative approach for evaluation of the antibody responses in patients with Lyme disease.Lyme disease is caused by the spirochete Borrelia burgdorferi, which is transmitted by the bite of a deer tick (Ixodes sp.) (24,29). One of the first signs of B. burgdorferi infection is erythema migrans (EM), a skin lesion that appears within a few days at the site of the bite. Subsequently, the spirochetes can disseminate into the bloodstream and then to various target tissues and cause neurological, cardiac, and rheumatological complications (24, 29). Some individuals develop post-Lyme disease syndrome (PLDS) and have lingering symptoms, such as fatigue, musculoskeletal pain, and cognitive impairment (22,24,29).Currently, the Centers for Diseases Control and Prevention (CDC) recommends the use of a two-tier approach for serological testing for Lyme disease (1). The two-tier approach includes an initial enzyme immunoassay or immunofluorescence assay, followed by Western blotting for positive or borderline samples. The limitations of the two-tier testing approach include a low sensitivity in the very early stages of the B. burgdorferi infection, subjectivity in the interpretation of the Western blot bands, and the significant amount of time and the significant cost for the process. Moreover, curre...

Multiplex Immunoassay for Lyme Disease Using VlsE1-IgG and pepC10-IgM Antibodies: Improving Test Performance through Bioinformatics

“…Given the complex nature of the host immune response to B. burgdorferi infection, the use of multiple serologic assays has been proposed to enhance either test sensitivity or specificity (2,6,12,17,34,35). Tests derived from continuous data generate binary (positive or negative) results when a cutoff value is chosen to achieve a desired specificity (e.g., 99%).…”

Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

“…If true, this finding would impact the determination of the background threshold setting for the assay, making it necessary to assign a distinct background threshold for each antigen-isotype combination. To test this hypothesis, we compiled a list from the literature of B. burgdorferi proteins that are known or hypothesized to be seroreactive in humans (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). From this list, a subset of 8 B. burgdorferi antigens was selected for further analysis in our assay due to their ability to be produced in large quantities as recombinant in-frame N-terminal glutathione S-transferase (GST) fusion proteins in E. coli.…”

“…This indicated that each antigen-isotype combination performed uniquely using the current iPCR protocol. These data provided the necessary parameters, including the mean background C q value and the standard deviation of that mean for determining an (24,37,38). The demonstration of iPCR equivalency to 2-tier testing using a panel of antigens led us to surmise that a more simplified version of the protocol using a single hybrid antigen was likely to be successful.…”

Synthetic Genomics and Synthetic Biology Applications Between Hopes and Concerns

“…Lyme disease) involving DNAsynthesis [42]. Rather complex DNA-synthesis and genomeassembly techniques have been used to generate entire viral genomes and to address the etiology and pathogenicity mechanisms of corresponding viruses, including the viruses that caused the 1918 influenza pandemic or that are responsible for SARS [43,44].…”