Development of enzyme-linked immunosorbent assays using recombinant borrelial antigens for serodiagnosis of Borrelia burgdorferi infection

Burkert, Sigrun; Rössler, Dieter; Münchhoff, Peter; Wilske, Bettina

doi:10.1007/s004300050014

Cited by 16 publications

(13 citation statements)

References 45 publications

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“…Furthermore, the administration of the OspA-based Lyme disease vaccine has complicated the application of ELISAs based upon WCL to identify B. burgdorferi infection in vaccinated individuals (1,26,78). Recent attempts have been made to identify single antigens or a combination of antigens that result in sensitivity similar to that generated against B. burgdorferi WCL but with an increase in the specificity of reactive antibodies (15,27,30,31,40,(42)(43)(44)58). In this study, a serological analysis of reactivity to the OspE-related, OspF-related, and Elp proteins was performed on patients with early and late Lyme disease, arbitrarily defined as less than 12 weeks p.i.…”

Section: Discussionmentioning

confidence: 99%

OspE-Related, OspF-Related, and Elp Lipoproteins Are Immunogenic in Baboons Experimentally Infected withBorrelia burgdorferiand in Human Lyme Disease Patients

Hefty¹,

Brooks²,

Jett³

et al. 2002

J Clin Microbiol

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Presently, the rhesus macaque is the only nonhuman primate animal model utilized for the study of Lyme disease. While this animal model closely mimics human disease, rhesus macaques can harbor the herpes B virus, which is often lethal to humans; macaques also do not express the full complement of immunoglobulin G (IgG) subclasses found in humans. Conversely, baboons contain the full complement of IgG subclasses and do not harbor the herpes B virus. For these reasons, baboons have been increasingly utilized as the basis for models of infectious diseases and studies assessing the safety and immunogenicity of new vaccines. Here we analyzed the capability of baboons to become infected with Borrelia burgdorferi, the agent of Lyme disease. Combined culture and PCR analyses of tick-and syringe-infected animals indicated that baboons are a sufficient host for B. burgdorferi. Analysis of the antibody responses in infected baboons over a 48-week period revealed that antibodies are generated early during infection against many borrelial antigens, including the various OspE, OspF, and Elp paralogs that are encoded on the ubiquitous 32-kb circular plasmids (cp32s). By using the baboon sera generated by experimental infection it was determined that a combination of two cp32-encoded lipoproteins, OspE and ElpB1, resulted in highly specific and sensitive detection of B. burgdorferi infection. An expanded analysis, which included 39 different human Lyme disease patients, revealed that a combination of the OspE and ElpB1 lipoproteins could be the basis for a new serodiagnostic assay for Lyme disease. Importantly, this novel serodiagnostic test would be useful independent of prior OspA vaccination status.Lyme disease, the most common arthropod-borne disease in North America (49,50,70), is a multisystem disorder characterized by dermatologic, cardiac, neurologic, and arthritic manifestations (68, 69). Lyme disease pathogenesis and Borrelia burgdorferi infectivity have been studied in numerous animal models; however, the disease manifestations observed vary widely among host species (9). The murine model of Lyme disease has been the most intensively investigated and is presently the preferred animal model for Lyme disease research. The mouse model has allowed researchers to gain valuable insight into the effects of various components of the immune system in relation to Lyme disease pathogenesis (10, 13, 57, 64, 68, 69). However, there are drawbacks to the mouse model of Lyme disease. For instance, the mouse immune system and how it responds to B. burgdorferi infection can differ from the human immune response to this organism. Furthermore, not all disease manifestations observed in humans also are observed in mice, especially the erythema migrans and neurologic symptoms typically associated with Lyme disease (74).Presently, the rhesus macaque (Macaca mulatta) provides the closest animal model to human Lyme disease. Infection of these animals with B. burgdorferi results in an almost complete spectrum of human disease and the various ...

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Section: Discussionmentioning

confidence: 99%

OspE-Related, OspF-Related, and Elp Lipoproteins Are Immunogenic in Baboons Experimentally Infected withBorrelia burgdorferiand in Human Lyme Disease Patients

Hefty¹,

Brooks²,

Jett³

et al. 2002

J Clin Microbiol

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“…The three recombinant p41/i-ELISAs were optimized and performed as described in detail by Burkert et al [6]. Briefly, microtiter plates were coated overnight with p41/i:B31, p41/i:PKo, and p41/i:PBi, respectively (2.5 µg/well).…”

Section: Recombinant P41/i-elisamentioning

confidence: 99%

“…However, the influence of the interspecies heterogeneity (among B. burgdorferi s. l.) of the internal flagellin region (p41/i) on serological assays for LB has only rarely been studied so far [6,42]. Therefore, the purpose of the current study was the comparative evaluation of six enzyme-linked immunosorbent assays (ELISAs) for the serodiagnosis of NB using (i) recombinant p41/i and (ii) wholecell detergent extracts of three strains representing the species pathogenic for humans in Europe.…”

Section: Introductionmentioning

confidence: 99%

Enzyme-linked immunosorbent assays with recombinant internal flagellin fragments derived from different species of Borrelia burgdorferi sensu lato for the serodiagnosis of Lyme neuroborreliosis

Hauser

Wilske²

1997

Medical Microbiology and Immunology

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The serodiagnosis of early Lyme neuroborreliosis is hampered by false negative results and one of the reasons could be the heterogeneity of strains of Borrelia burgdorferi sensu lato. For this study IgG enzyme-linked immunosorbent assays (ELISAs) with recombinant internal flagellin fragments (p41/i; amino acids 129-251) derived from strains PKo (B. afzelii), B31 (B. burgdorferi sensu stricto), and PBi (B. garinii) and ELISAs with whole-cell detergent extracts of strains PKo, PKa2 (B. burgdorferi sensu stricto), and PBi were compared. Cut off absorbance values were defined by the 94th and 92th percentiles of 200 sera from blood donors. Sera from patients with clinically defined neuroborreliosis [n = 88; 41 culture proven and 47 intentionally selected by the criteria specific IgG cerebrospinal fluid/serum index > or = 2 and a serum IgG immunofluorescence assay value of < 1:64] were tested. The sensitivities of the three whole-cell detergent-extract ELISAs were similar (46.6-49.2%), whereas those of the recombinant ELISAs were highest with p41/i:PBi (57.1%) and lowest with p41/i:PKo (21.5%). A combination of the p41/i:PBi and the p41/i:B31 test was most sensitive (59.8%). The correlation of absorbance values of different assays was very high for the three whole-cell detergent-extract ELISAs, whereas the absorbance values obtained with the three recombinant tests differed considerably. The greatest differences were observed between p41/i:PKo and any of the other two recombinant antigens. The differences in immune reactivity of patients sera (due to strain heterogeneity?) seems to have more influence on the results of an assay based on a single antigen than on a whole-cell-based test.

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“…Several recombinant borrelial antigens (OspA, OspB, OspC, OspE, OspF, p22, BmpA, BBK32, BBK50, VlsE, p100, 14-kDa internal flagellin fragment) (5,7,16,21,22,24,25,31,35) and chimeric borrelial proteins OspA, OspB, OspC, flagellin (p41), and p93 (13) have been studied to improve serologic diagnosis. Of these proteins, BmpA (32) and OspC (8,26,27,30,31) have been suggested as antigens which induce early IgM responses.…”

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confidence: 99%

Recombinant Flagellin A Proteins from Borrelia burgdorferi Sensu Stricto, B . afzelii , and B. garinii in Serodiagnosis of Lyme Borreliosis

Panelius¹,

Lahdenne

Saxén

et al. 2001

J Clin Microbiol

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Genes for flagellin A (FlaA) proteins from European borrelial strains of Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii were cloned and sequenced. An identity of 92 to 93% was observed in the flaA sequences of the different species. Polyhistidine-tagged recombinant FlaA (rFlaA) proteins were produced in Escherichia coli and used as antigens in Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). In immunoglobulin G (IgG) WB, 71% (10 of 14) of the sera from neuroborreliosis and 86% (12 of 14) of those from Lyme arthritis patients reacted with one to three rFlaAs. In IgG ELISA, 74% (14 of 19) and 79% (15 of 19) of patients with neuroborreliosis and arthritis, respectively, were positive. The immunoreactivity in local European patient sera was stronger against rFlaA from B. garinii and B. afzelii than against rFlaA from B. burgdorferi sensu stricto. Neither IgG nor IgM ELISA was sensitive in the serodiagnosis of erythema migrans. Serum samples from patients with syphilis and systemic lupus erythematosus showed mild cross-reactivity in IgG tests. Sera from Yersinia enterocolitica or beta-hemolytic Streptococcus infections showed only occasional responses. With IgM ELISA, 58% (11 of 19) and 37% (7 of 19) of patients with neuroborreliosis and arthritis, respectively, were positive. Cross-reactive antibodies to FlaA, especially in serum samples from patients with rheumatoid factor positivity and Epstein-Barr virus infection, reduced the specificity of IgM serodiagnosis. Therefore, rFlaA seems to have a limited role for IgM serodiagnosis, yet rFlaA might be useful in the IgG serodiagnosis of disseminated Lyme borreliosis.Laboratory diagnosis of Lyme borreliosis (LB) is mainly based on serology, although the present serologic tests have unsatisfactory sensitivity and specificity (34). Routine laboratory testing uses enzyme-linked immunosorbent assays (ELISA) with borrelial whole-cell lysate (WCL) or flagella (consisting mainly of polymerized FlaB protein) as the most commonly used antigens. A two-step approach with ELISA followed by a confirmatory Western blot (WB) has been recommended for positive or borderline results (19,36). Especially in Europe, the applicability of this procedure has, however, remained doubtful (3, 14) because three or more borrelial species cause LB (38). Several difficulties complicate LB serology. Firstly, immunoglobulin G (IgG) antibody responses are often delayed during the early stages of LB. Even at late-stage LB, 5 to 10% of patients do not have elevated antibody levels (29), perhaps due to diversion of the host immune response towards Th1 immunity by borrelial factors (17). Secondly, viral infections cause false-positive IgM results in several LB tests (4). Thirdly, in a subgroup of patients antibody levels may stay high after successful treatment of LB even for prolonged periods (6, 15).Several recombinant borrelial antigens (OspA, OspB, OspC, OspE, OspF, p22, BmpA, BBK32, BBK50, VlsE, p100, 14-kDa internal flagellin fragment) (5,7,16,21,22,24,25,31,35) and...

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Development of enzyme-linked immunosorbent assays using recombinant borrelial antigens for serodiagnosis of Borrelia burgdorferi infection

Cited by 16 publications

References 45 publications

OspE-Related, OspF-Related, and Elp Lipoproteins Are Immunogenic in Baboons Experimentally Infected withBorrelia burgdorferiand in Human Lyme Disease Patients

OspE-Related, OspF-Related, and Elp Lipoproteins Are Immunogenic in Baboons Experimentally Infected withBorrelia burgdorferiand in Human Lyme Disease Patients

Enzyme-linked immunosorbent assays with recombinant internal flagellin fragments derived from different species of Borrelia burgdorferi sensu lato for the serodiagnosis of Lyme neuroborreliosis

Recombinant Flagellin A Proteins from Borrelia burgdorferi Sensu Stricto, B . afzelii , and B. garinii in Serodiagnosis of Lyme Borreliosis

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