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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 bead-based 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.

SUBMITTER: Embers ME

PROVIDER: S-EPMC4820514 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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

Embers Monica E ME Hasenkampf Nicole R NR Barnes Mary B MB Didier Elizabeth S ES Philipp Mario T MT Tardo Amanda C AC

Clinical and vaccine immunology : CVI 20160404 4

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 f ...[more]

PMID: 26843487

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Project description:BackgroundFree-ranging chickens are often infected with Toxoplasma gondii and seroconvert upon infection. This indicates environmental contamination with T. gondii.MethodsHere, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1bio) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. Serum antibodies were detected by a fluorophore-coupled secondary antibody. Beads of differing color codes were conjugated with anti-chicken IgY or chicken serum albumin and served for each sample as an internal positive or negative control, respectively. The assay was validated with sera from experimentally and naturally infected chickens. The results were compared to those from reference methods, including other serological tests, PCRs and bioassay in mice.ResultsIn experimentally infected chickens, the vast majority (98.5%, n = 65/66) of birds tested seropositive in the BBMA. This included all chickens positive by magnetic-capture PCR (100%, n = 45/45). Most, but not all inoculated and TgSAG1bio-BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs (TgSAG1-ELISASL, n = 61/65; or TgSAG1-ELISASH, n = 60/65), or positive in an immunofluorescence assay (IFAT, n = 64/65) and in a modified agglutination test (MAT, n = 61/65). All non-inoculated control animals (n = 28/28, 100%) tested negative. In naturally exposed chickens, the TgSAG1bio-BBMA showed a high sensitivity (98.5%; 95% confidence interval, CI: 90.7-99.9%) and specificity (100%; 95% CI: 85.0-100%) relative to a reference standard established using ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in bioassay or by PCR tested positive in the TgSAG1bio-BBMA (93.5%; 95% CI: 77.1-98.9%), while all bioassay- or PCR-negative chickens remained negative (100%; 95% CI: 85.0-100%).ConclusionsThe TgSAG1bio-BBMA represents a suitable method for the detection of T. gondii infections in chickens with high sensitivity and specificity, which is comparable or even superior to other tests. Since assays based on this methodology allow for the simultaneous analysis of a single biological sample with respect to multiple analytes, the described assay may represent a component in future multiplex assays for broad serological monitoring of poultry and other farm animals for various pathogens.

Dataset Information

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

Publications

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

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