Five-Antigen Fluorescent Bead-Based Assay for Diagnosis of Lyme Disease

Abstract

The systematically difficult task of diagnosing Lyme disease can be simplified by sensitive and specific laboratory tests. The currently recommended two-tier test for serology is highly specific but falls short in sensitivity, especially in the early acute phase. We previously examined serially collected serum samples from Borrelia burgdorferi-infected rhesus macaques and defined a combination of antigens that could be utilized for detection of infection at all phases of disease in humans. The five B. burgdorferi antigens, consisting of OspC, OspA, DbpA, OppA2, and the C6 peptide, were combined into a fluorescent cytometric beadbased assay for the detection of B. burgdorferi antigen-specific IgG antibodies. Samples from Lyme disease patients and controls were used to determine the diagnostic value of this assay. Using this sample set, we found that our five-antigen multiplex IgG assay exhibited higher sensitivity (79.5%) than the enzyme immunoassay (EIA) (76.1%), the two-tier test (61.4%), and the C6 peptide enzyme-linked immunosorbent assay (ELISA) (77.2%) while maintaining specificity over 90%. When detection of IgM was added to the bead-based assay, the sensitivity improved to 91%, but at a cost of reduced specificity (78%). These results indicate that the rational combination of antigens in our multiplex assay may offer an improved serodiagnostic test for Lyme disease. With an estimated 300,000 new cases each year, Lyme disease is the most common vector-borne disease in North America (http://www.cdc.gov/lyme/stats/humanCases.html) (1). The burden of this disease in many parts of Europe is also staggering (1, 2). Treatment with antibiotics is generally effective, even more so when employed soon after infection (3). Therefore, early and reliable laboratory diagnosis is critical for effective cure of Lyme disease patients.Patient serum antibodies specific for Borrelia burgdorferi antigens are detected with currently recommended laboratory tests for Lyme disease. While the detection of antigen may be preferred for early diagnosis, this is encumbered by the absence of detectable spirochetes or spirochetal antigen in the bloodstream once the organism has disseminated. Thus, the use of antigen detection from a blood or skin biopsy specimen has not demonstrated favorable sensitivity (4). The two most commonly used tests for diagnosis of Lyme disease in North America are (i) the two-tier test that includes an enzyme-linked immunosorbent assay (ELISA) and confirmatory Western blotting and (ii) the C6 test, where antibodies to a specific peptide within a conserved region of VlsE, the B. burgdorferi antigen, are detected (5-8). Both the twotier and C6 tests exhibit high specificity and are most sensitive for patients in the disseminated phases of disease (5, 8-10). In addition, the C6 test has been evaluated as an indicator of treatment outcome in the United States (11, 12), with a Ն4-fold decline in antibody titer (up to 6 months after treatment) in a majority of patients. The Western blot procedure is technically dema...

Variable manifestations, diverse seroreactivity and post-treatment persistence in non-human primates exposed to Borrelia burgdorferi by tick feeding

“…The responses to five B . burgdorferi antigens were measured using a Luminex ® -based assay developed by our laboratory [ 40 ]. As expected, the C6 antibody levels waned after antibiotic treatment in those animals that received doxycycline at 16 weeks post-inoculation ( Fig 3 ).…”

“…Longitudinal antibody responses targeting five different B . burgdorferi antigens (OspA, OspC, DbpA, OppA-2 and the C6 peptide [ 39 ]) were measured by a recently-described Luminex ® -based assay [ 40 ]. Samples from each time point were analyzed in triplicate.…”

A multiplex serologic platform for diagnosis of tick-borne diseases

“…This strategy improves assay specificity by eliminating non-specific and potentially cross-reactive epitopes. Targeting a combination of such antigens can enhance assay sensitivity and has been shown to improve the diagnosis of early Lyme disease 32 – 34 . Employing this approach, we established the first highly-multiplexed, array-based assay for diagnostic serology of TBDs called the Tick-borne disease Serochip (TBD-Serochip).…”

A fluorescent plasmonic biochip assay for multiplex screening of diagnostic serum antibody targets in human Lyme disease

“…Important considerations when pursuing indirect detection include achieving high analytical sensitivity, as well as being able to distinguish between treated and active disease. A multiplexed and quantitative strategy may be particularly useful for screening diagnostic targets (Table 1), understanding the individualized immune response to LD infection, and capturing the kinetics of this response during the course of disease and treatment [31]. Here we describe a protein microarray, in the form of a compact biochip, that can be analyzed with high sensitivity using grating-coupled fluorescence plasmonics (GC-FP) technology.…”