scholarly journals Evaluation of Selected Borrelia burgdorferi lp54 Plasmid-Encoded Gene Products Expressed during Mammalian Infection as Antigens To Improve Serodiagnostic Testing for Early Lyme Disease

2015 ◽  
Vol 22 (11) ◽  
pp. 1176-1186 ◽  
Author(s):  
Zachary P. Weiner ◽  
Rebecca M. Crew ◽  
Kevin S. Brandt ◽  
Amy J. Ullmann ◽  
Martin E. Schriefer ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Recombinant Proteins ◽  
Erythema Migrans ◽  
Disease Patient ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Early Stages ◽  
Lyme Carditis ◽  
Mammalian Infection

ABSTRACTLaboratory testing for the diagnosis of Lyme disease is performed primarily by serologic assays and is accurate for detection beyond the acute stage of the infection. Serodiagnostic assays to detect the early stages of infection, however, are limited in their sensitivity, and improvement is warranted. We analyzed a series ofBorrelia burgdorferiproteins known to be induced within feeding ticks and/or during mammalian infection for their utility as serodiagnostic markers against a comprehensive panel of Lyme disease patient serum samples. The antigens were assayed for IgM and IgG reactivity in line immunoblots and separately by enzyme-linked immunosorbent assay (ELISA), with a focus on reactivity against early Lyme disease with erythema migrans (EM), early disseminated Lyme neuroborreliosis, and early Lyme carditis patient serum samples. By IgM immunoblotting, we found that recombinant proteins BBA65, BBA70, and BBA73 reacted with early Lyme EM samples at levels comparable to those of the OspC antigen used in the current IgM blotting criteria. Additionally, these proteins reacted with serum samples from patients with early neuroborreliosis and early carditis, suggesting value in detecting early stages of this disease progression. We also found serological reactivity against recombinant proteins BBA69 and BBA73 with early-Lyme-disease samples using IgG immunoblotting and ELISA. Significantly, some samples that had been scored negative by the Centers for Disease Control and Prevention-recommended 2-tiered testing algorithm demonstrated positive reactivity to one or more of the antigens by IgM/IgG immunoblot and ELISA. These results suggest that incorporating additionalin vivo-expressed antigens into the current IgM/IgG immunoblotting tier in a recombinant protein platform assay may improve the performance of early-Lyme-disease serologic testing.

scholarly journals Outer Surface Protein C Peptide Derived from Borrelia burgdorferi Sensu Stricto as a Target for Serodiagnosis of Early Lyme Disease

2013 ◽  
Vol 20 (4) ◽  
pp. 474-481 ◽  
Author(s):  
Paul M. Arnaboldi ◽  
Rudra Seedarnee ◽  
Mariya Sambir ◽  
Steven M. Callister ◽  
Josephine A. Imparato ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Bacterial Species ◽  
Erythema Migrans ◽  
Early Infection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Attractive Alternative ◽  
Serum Samples ◽  
Peptide Epitope ◽  
Content Type

ABSTRACTCurrent 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 forBorrelia burgdorferias diagnostic targets are an attractive alternative to current assays. Using an overlapping peptide library, we mapped linear epitopes in OspC, a critical virulence factor ofB. burgdorferirequired 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.

scholarly journals Human Antibody Responses to VlsE Antigenic Variation Protein of Borrelia burgdorferi

1999 ◽  
Vol 37 (12) ◽  
pp. 3997-4004 ◽  
Author(s):  
M. B. Lawrenz ◽  
J. M. Hardham ◽  
R. T. Owens ◽  
J. Nowakowski ◽  
A. C. Steere ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Lupus Erythematosus ◽  
Antigenic Variation ◽  
Erythema Migrans ◽  
Disease Patient ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Antibody ◽  
Whole Cell ◽  
Cell Elisa

VlsE is a 35-kDa surface-exposed lipoprotein of Borrelia burgdorferi that was shown previously to undergo antigenic variation through segmental recombination of silent vlscassettes with vlsE during experimental mouse infections. Previous data had indicated that sera from North American Lyme disease patients and experimentally infected animals contained antibodies reactive with VlsE. In this study, sera from patients with Lyme disease, syphilis, and autoimmune conditions as well as from healthy controls were examined for reactivity with VlsE by Western blotting and enzyme-linked immunosorbent assay (ELISA). Strong Western blot reactivity to a recombinant VlsE cassette region protein was obtained consistently with Lyme disease sera. Although sera from Lyme disease patients also reacted with a band corresponding to VlsE in B. burgdorferi B31-5A3, interpretation was complicated by low levels of VlsE expression in in vitro-cultured B. burgdorferi and by the presence of comigrating bands. An ELISA using recombinant VlsE was compared with an ELISA using sonically disrupted B. burgdorferi as the antigen. For a total of 93 Lyme disease patient sera examined, the VlsE ELISA yielded sensitivities of 63% for culture-confirmed erythema migrans cases and 92% for later stages, as compared to 61 and 98%, respectively, for the “whole-cell” ELISA. The specificities of the two assays with healthy blood donor sera were comparable, but the VlsE ELISA was 90% specific with sera from syphilis patients, compared to 20% specificity for the whole-cell ELISA with this group. Neither assay showed reactivity with a panel of sera from 20 non-Lyme disease arthritis patients or 20 systemic lupus erythematosus patients. Our results indicate that VlsE may be useful in the immunodiagnosis of Lyme disease and may offer greater specificity than ELISAs using whole B. burgdorferi as the antigen.

scholarly journals The Borrelia burgdorferi 37-Kilodalton Immunoblot Band (P37) Used in Serodiagnosis of Early Lyme Disease Is the flaA Gene Product

1999 ◽  
Vol 37 (3) ◽  
pp. 548-552 ◽  
Author(s):  
Robert D. Gilmore ◽  
Rendi L. Murphree ◽  
Angela M. James ◽  
Sarah A. Sullivan ◽  
Barbara J. B. Johnson
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Genomic Library ◽  
Disease Patient ◽  
Humoral Response ◽  
Immunoglobulin M ◽  
Leader Peptide ◽  
Serum Samples ◽  
Present Generation ◽  
Periplasmic Flagella

The 37-kDa protein (P37) of Borrelia burgdorferi is an antigen that elicits an early immunoglobulin M (IgM) antibody response in Lyme disease patients. The P37 gene was cloned from aB. burgdorferi genomic library by screening with antibody from a Lyme disease patient who had developed a prominent humoral response to the P37 antigen. DNA sequence analysis of this clone revealed the identity of P37 to be FlaA, an outer sheath protein of the periplasmic flagella. Recombinant P37 expression was accomplished inEscherichia coli by using a gene construct with the leader peptide deleted and fused to a 38-kDa E. coli protein. The recombinant antigen was reactive in IgM immunoblots using serum samples from patients clinically diagnosed with early Lyme disease that had been scored positive for B. burgdorferi anti-P37 reactivity. Lyme disease patient samples serologically negative for theB. burgdorferi P37 protein did not react with the recombinant. Recombinant P37 may be a useful component of a set of defined antigens for the serodiagnosis of early Lyme disease. This protein can be utilized as a marker in diagnostic immunoblots, aiding in the standardization of the present generation of IgM serologic tests.

scholarly journals Impact of Clinical Variables on Borrelia burgdorferi-Specific Antibody Seropositivity in Acute-Phase Sera from Patients in North America with Culture-Confirmed Early Lyme Disease

2008 ◽  
Vol 15 (10) ◽  
pp. 1519-1522 ◽  
Author(s):  
Gary P. Wormser ◽  
John Nowakowski ◽  
Robert B. Nadelman ◽  
Paul Visintainer ◽  
Andrew Levin ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Acute Phase ◽  
Erythema Migrans ◽  
Skin Lesions ◽  
Enzyme Linked Immunosorbent Assay ◽  
Clinical Variables ◽  
Gender And Age ◽  
Serologic Assay ◽  
Single Lesion

ABSTRACT Erythema migrans, the most common manifestation of Lyme disease, has been associated with highly variable rates of seropositivity for antibodies to Borrelia burgdorferi. Differences in the sensitivities of serologic assays for the detection of these antibodies, however, may not be the only or even the primary explanation for this observation. We investigated the impacts of four clinical variables on seropositivity—the duration of erythema migrans, the presence of single versus multiple skin lesions, and the gender and age of the patient. In this analysis, three different serologic tests were performed on acute-phase sera from 175 untreated patients with culture-confirmed erythema migrans: the C6 single-peptide enzyme-linked immunosorbent assay (ELISA), a commercially available ELISA in which a whole-cell sonicate of B. burgdorferi was the antigen, and a two-tier procedure. Irrespective of the serologic test performed, the results showed that seropositivity rates increased with the duration of the erythema migrans for patients with single lesions (P < 0.001) but not for those with multiple skin lesions. The variability in seropositivity rates was greatest for the two-tier testing strategy, with a >6-fold-higher rate of seropositivity among patients with a single lesion of 22- to 30-day duration than among those whose skin lesion was of 1- to 7-day duration (85.7 versus 14.1%; P < 0.001). Rates of seropositivity by each of the testing methods were also significantly higher for patients with multiple skin lesions than for those with single lesions (P < 0.001). In contrast, seropositivity rates were not affected by either the gender or the age of the patient. Thus, in patients with erythema migrans, certain clinical variables such as the duration and number of skin lesions had a profound impact on seropositivity rates, irrespective of the serologic assay performed.

Comparative evaluation of three different ELISA methods for the diagnosis of early culture-confirmed Lyme disease in Italy

2005 ◽  
Vol 54 (4) ◽  
pp. 361-367 ◽  
Author(s):  
Antonella Marangoni ◽  
Monica Sparacino ◽  
Francesca Cavrini ◽  
Elisa Storni ◽  
Valeria Mondardini ◽  
...  
Keyword(s):  
Immune Response ◽  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
False Positive ◽  
Comparative Evaluation ◽  
Endemic Area ◽  
Blood Donors ◽  
Erythema Migrans ◽  
Serum Samples ◽  
Positive Reactivity

In this study the raising and development of the immune response to Borrelia burgdorferi infection in 45 Italian patients suffering from culture-confirmed Lyme borreliosis erythema migrans was investigated. A total of 95 serially collected serum samples were tested by using three different commercial ELISAs: recomWell Borrelia (Mikrogen), Enzygnost Borreliosis (DADE Behring) and Quick ELISA C6 Borrelia (Immunetics). The sensitivities of the ELISAs were as follows: Enzygnost Borreliosis IgM, 70.5 %; Quick ELISA C6 Borrelia, 62.1 %; recomWell Borrelia IgM, 55.7 %; recomWell Borrelia IgG, 57.9 %; and Enzygnost Borreliosis IgG, 36.8 %. In order to compare the specificity values of the three ELISAs, a panel of sera obtained from blood donors (210 samples coming from a non-endemic area and 24 samples from an endemic area) was tested, as well as sera from patients suffering from some of the most common biological conditions that could result in false-positive reactivity in Lyme disease serology (n = 40). RecomWell Borrelia IgG and recomWell Borrelia IgM were the most specific (97.1 % and 98.9 %, respectively), followed by Quick ELISA C6 Borrelia (96.7 %). Enzygnost Borreliosis IgG and IgM achieved 90.1 % and 92.3 % specificity, respectively. Sera that gave discrepant results when tested by the three ELISAs were further analysed by Western blotting.

scholarly journals Epitope Length, Genospecies Dependency, and Serum Panel Effect in the IR6 Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Borrelia burgdorferi

2007 ◽  
Vol 14 (7) ◽  
pp. 875-879 ◽  
Author(s):  
Maria J. C. Gomes-Solecki ◽  
Luciana Meirelles ◽  
John Glass ◽  
Raymond J. Dattwyler
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Immunosorbent Assay ◽  
Erythema Migrans ◽  
The United States ◽  
Enzyme Linked Immunosorbent Assay ◽  
Diagnostic Assay ◽  
Assay Sensitivity ◽  
Selection Of ◽  
Antigenic Epitopes

ABSTRACTIn the absence of erythema migrans, the basis for diagnosis of Lyme disease is the demonstration of an antibody response againstBorrelia burgdorferiin an appropriate clinical setting. The C6 enzyme-linked immunosorbent assay, based on the IR6 region of VlsE, has become widely used in both the United States and Europe. We mapped the antigenic epitopes of IR6 to a shorter sequence that is equivalent in sensitivity and specificity to the full-length IR6 25-residue peptide. In addition, we observed significant differences in sensitivity between serum panels (60 to 100%), indicating that the selection of the serum panels can shape the apparent overall sensitivity of the assay. Contrary to prior reports, the assay sensitivity is greater when the IR6 peptide is derived from the sequence of the same infectingBorreliagenospecies. Using our North American panels and the two panels obtained from European Lyme disease patients, we determined that the IR6 assay that is based on a single genospecies ofBorreliaspp. is not optimal for use as a universal diagnostic assay for Lyme disease.

scholarly journals Borrelia burgdorferi Spirochetes That Harbor Only a Portion of the lp28-1 Plasmid Elicit Antibody Responses Detectable with the C6 Test for Lyme Disease

2006 ◽  
Vol 14 (1) ◽  
pp. 90-93 ◽  
Author(s):  
Monica E. Embers ◽  
Gary P. Wormser ◽  
Ira Schwartz ◽  
Dale S. Martin ◽  
Mario T. Philipp
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Antibody Responses ◽  
Antibody Detection ◽  
23S Rrna ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Altered Expression ◽  
Genetic Region ◽  
Length Polymorphisms

ABSTRACT Detection of antibody to C6, a peptide that reproduces the sequence of the sixth invariable region within the central domain of the VlsE protein of Borrelia burgdorferi, is used currently for the serologic diagnosis of Lyme disease in humans. B. burgdorferi isolates taken from infected humans can be categorized into specific genetic subtypes (designated RST1, -2, and -3) by restriction fragment length polymorphisms in the 16S to 23S rRNA spacer sequence. Many of these, usually categorized as RST2, retain only segments of the linear plasmid lp28-1, which encodes VlsE. The VlsE genetic region is retained, but altered expression of this molecule could affect diagnosis by the C6 enzyme-linked immunosorbent assay (ELISA). Serum samples from patients infected with each of the three genotypes and from mice infected with three RST2 isolates were tested with the C6 ELISA. Such isolates elicited marked C6 responses in infected mice. The sensitivity of C6 antibody detection in patients infected with RST2 spirochetes was statistically indistinguishable from detection of RST1 and RST3 infections. These findings demonstrate that diagnosis by C6 ELISA remains effective for infection with all B. burgdorferi genotypes, including those with incomplete lp28-1 plasmids.

scholarly journals Enzyme-Linked Immunosorbent Assay Using Recombinant OspC and the Internal 14-kDa Flagellin Fragment for Serodiagnosis of Early Lyme Disease

1998 ◽  
Vol 36 (4) ◽  
pp. 857-861 ◽  
Author(s):  
Sebastian Rauer ◽  
Nicole Spohn ◽  
Christiane Rasiah ◽  
Uwe Neubert ◽  
Arnold Vogt
Keyword(s):  
Lyme Disease ◽  
Immunosorbent Assay ◽  
Erythema Migrans ◽  
Cell Extract ◽  
Sensu Stricto ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Epstein Barr Virus Infection ◽  
Serum Samples ◽  
Antigen Elisa

The outer surface protein C (OspC) and the internal 14-kDa flagellin fragment of strain GeHo of Borrelia burgdorferisensu stricto were expressed as recombinant proteins inEscherichia coli and were purified for use in an immunoglobulin M (IgM) enzyme-linked immunosorbent assay (OspC–14-kDa antigen ELISA). No hint at disturbing protein-protein interferences, which might influence the availability of immunoreactive epitopes, was found when the recombinant antigens were combined in the ELISA. The recombinant OspC–14-kDa antigen ELISA was compared to a commercial IgM ELISA that used a detergent cell extract from Borrelia afzelii PKo as the antigen. According to the manufacturer’s information, the cell extract contains, in addition to other antigens, the following diagnostically relevant antigens: the 100-kDa (synonyms, 93- and 83-kDa antigens), 41-kDa, OspA, OspC, and 17-kDa antigens. The specificity was adjusted to 95% on the basis of data for 154 healthy controls. On testing of 104 serum samples from patients with erythema migrans (EM), the sensitivity of the recombinant ELISA (46%) for IgM antibodies was similar to that of the commercial ELISA (45%). However, when 42 serum samples from patients with polyclonal B-cell stimulation due to an Epstein-Barr virus infection were tested, false-positive reactions were significantly less frequent in the recombinant ELISA (10%) than in the whole-cell-extract ELISA (23%). OspC displays sequence heterogeneity of up to 40% according to the genomospecies. However, when the reactions of serum specimens from controls and EM patients with OspC from representative strains of B. burgdorferi sensu stricto (strain GeHo) and B. afzelii (strain PKo) were compared in an ELISA, almost no differences in specificity and sensitivity were seen. This demonstrates that the sera predominantly recognize the common epitopes of OspC tested in this study. In conclusion, we suggest that the OspC–14-kDa antigens ELISA is a suitable test for the detection of an IgM response in early Lyme disease.

scholarly journals Sensitive and Specific Serodiagnosis of Lyme Disease by Enzyme-Linked Immunosorbent Assay with a Peptide Based on an Immunodominant Conserved Region of Borrelia burgdorferi VlsE

1999 ◽  
Vol 37 (12) ◽  
pp. 3990-3996 ◽  
Author(s):  
Fang Ting Liang ◽  
Allen C. Steere ◽  
Adriana R. Marques ◽  
Barbara J. B. Johnson ◽  
James N. Miller ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Immunosorbent Assay ◽  
Synthetic Peptide ◽  
Late Phase ◽  
Hospitalized Patients ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Variable Surface ◽  
Conserved Region

VlsE, the variable surface antigen of Borrelia burgdorferi, contains an immunodominant conserved region named IR6. In the present study, the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on a 26-mer synthetic peptide (C6) with the IR6 sequence was explored. Sensitivity was assessed with serum samples (n = 210) collected from patients with clinically defined Lyme disease at the acute (early localized or early disseminated disease), convalescent, or late disease phase. The sensitivities for acute-, convalescent-, and late-phase specimens were 74% (29 of 39), 85 to 90% (34 of 40 to 35 of 39), and 100% (59 of 59), respectively. Serum specimens from early neuroborreliosis patients were 95% positive (19 of 20), and those from an additional group of patients with posttreatment Lyme disease syndrome yielded a sensitivity of 62% (8 of 13). To assess the specificity of the peptide ELISA, 77 serum samples from patients with other spirochetal or chronic infections, autoimmune diseases, or neurologic diseases and 99 serum specimens from hospitalized patients in an area where Lyme disease is not endemic were examined. Only two potential false positives from the hospitalized patients were found, and the overall specificity was 99% (174 of 176). Precision, which was assessed with a panel of positive and negative serum specimens arranged in blinded duplicates, was 100%. Four serum samples with very high anti-OspA antibody titers obtained from four monkeys given the OspA vaccine did not react with the C6 peptide. This simple, sensitive, specific, and precise ELISA may contribute to alleviate some of the remaining problems in Lyme disease serodiagnosis. Because of its synthetic peptide base, it will be inexpensive to manufacture. It also will be applicable to serum specimens from OspA-vaccinated subjects.

scholarly journals Characterization of a Borrelia burgdorferi VlsE Invariable Region Useful in Canine Lyme Disease Serodiagnosis by Enzyme-Linked Immunosorbent Assay

2000 ◽  
Vol 38 (11) ◽  
pp. 4160-4166 ◽  
Author(s):  
Fang Ting Liang ◽  
Richard H. Jacobson ◽  
Reinhard K. Straubinger ◽  
Amy Grooters ◽  
Mario T. Philipp
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Antibody Response ◽  
Immunosorbent Assay ◽  
Protein A ◽  
Immunoblot Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Entire Study ◽  
Variable Surface

Sera collected from dogs experimentally infected withBorrelia burgdorferi by tick inoculation were analyzed for an antibody response to each of the six invariable regions (IRs; i.e., IR1 to IR6) of VlsE, the variable surface antigen of B. burgdorferi. Six synthetic peptides (C1 to C6), which reproduced the six IR sequences were used as peptide-based, enzyme-linked immunosorbent assay (ELISA) antigens. Two IRs, IR2 and IR6, were found to be immunodominant. Studies with serially collected serum samples from experimentally infected dogs revealed that the antibody response to IR6 appears earlier and is stronger than that to IR2. Thus, the IR6 sequence alone appeared to be sufficient for serodiagnosis. When C6 alone was used as antigen, the peptide-based ELISA was positive in 7 of 23 dogs (30%) as early as 3 weeks postinfection. All dogs (n = 33) became strongly positive 1 or 2 weeks later, and this response persisted for the entire study, which lasted for 69 weeks. Of 55 sera submitted by veterinarians from dogs suspected of having Lyme disease, 19 were also positive by the C6 ELISA, compared to 20 positives detected by immunoblot analysis using cultured B. burgdorferi lysates as antigen. The sensitivity of using C2 and C6 together for detecting specific antibody in both experimentally infected and clinically diagnosed dogs was not better than sensitivity with C6 alone, confirming that C6 suffices as a diagnostic probe. Moreover, the C6 ELISA yielded 100% specificity with serum samples collected from 70 healthy dogs, 14 dogs with infections other than B. burgdorferi, and 15 animals vaccinated with either outer surface protein A, whole-spirochete vaccines, or the common puppy-vaccines. Therefore, this C6 ELISA was both sensitive and specific for the serodiagnosis of canine Lyme disease and could be used with vaccinated dogs.

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