scholarly journals Immunoproteomic analysis of Borrelia miyamotoi for the identification of serodiagnostic antigens

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Emma K. Harris ◽  
Marisa R. Harton ◽  
Maria Angela de Mello Marques ◽  
John T. Belisle ◽  
Claudia R. Molins ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Specific Antigen ◽  
Disease Patient ◽  
Borrelia Miyamotoi ◽  
Serum Samples ◽  
Human Patient ◽  
Highly Sensitive ◽  
Associated Proteins ◽  
Multiple Variable ◽  
Health Significance

AbstractThe tick-borne spirochete, Borrelia miyamotoi, is an emerging pathogen of public health significance. Current B. miyamotoi serodiagnostic testing depends on reactivity against GlpQ which is not highly sensitive on acute phase serum samples. Additionally, anti-B. miyamotoi antibodies can cross-react with C6 antigen testing for B. burgdorferi, the causative agent of Lyme disease, underscoring the need for improved serological assays that produce accurate diagnostic results. We performed an immunoproteomics analysis of B. miyamotoi proteins to identify novel serodiagnostic antigens. Sera from mice infected with B. miyamotoi by subcutaneous inoculation or tick bite were collected for immunoblotting against B. miyamotoi membrane-associated proteins separated by 2-dimensional electrophoresis (2DE). In total, 88 proteins in 40 2DE immunoreactive spots were identified via mass spectrometry. Multiple variable large proteins (Vlps) and a putative lipoprotein were among those identified and analyzed. Reactivity of anti-B. miyamotoi sera against recombinant Vlps and the putative lipoprotein confirmed their immunogenicity. Mouse anti-B. burgdorferi serum was cross-reactive to all recombinant Vlps, but not against the putative lipoprotein by IgG. Furthermore, antibodies against the recombinant putative lipoprotein were present in serum from a B. miyamotoi-infected human patient, but not a Lyme disease patient. Results presented here provide a comprehensive profile of B. miyamotoi antigens that induce the host immune response and identify a putative lipoprotein as a potentially specific antigen for B. miyamotoi serodetection.

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 Evidence That the Variable Regions of the Central Domain of VlsE Are Antigenic during Infection with Lyme Disease Spirochetes

2002 ◽  
Vol 70 (8) ◽  
pp. 4196-4203 ◽  
Author(s):  
John V. McDowell ◽  
Shian-Ying Sung ◽  
Linden T. Hu ◽  
Richard T. Marconi
Keyword(s):  
Lyme Disease ◽  
Immune Evasion ◽  
Antigenic Variation ◽  
Protein A ◽  
Disease Patient ◽  
Serum Samples ◽  
Variable Regions ◽  
Central Domain ◽  
Humoral Immune ◽  
Antigenic Properties

ABSTRACT It has been postulated that the vls system of the Lyme disease spirochetes contributes to immune evasion through antigenic variation. While it is clear that vlsE undergoes sequence change within its variable regions at a high frequency during the early stages of infection, a definitive role in immune evasion has not been demonstrated. In this report we assessed the murine and human humoral immune response to recombinant (r)-VlsE variants that originally arose during infection in mice. Immunoblot analyses of r-VlsE variants were conducted by using serum samples collected from mice infected with Borrelia burgdorferi clones that carried different vlsE variants. All of the r-VlsE variants were recognized by infection sera regardless of the identity of the infecting clone or isolate. In addition, all variants were immunoreactive with a panel of human Lyme disease patient serum samples. It is evident from these analyses that the infection-induced VlsE variants share common epitopes that reside within conserved segments of these proteins. However, preabsorption experiments revealed that the variable regions of the central domain of VlsE, which undergo rapid mutation during infection, also influence the antigenic properties of the protein. A subset of the antibodies elicited against vlsE variants that differ in the sequences of their variable regions were found to be variant specific. Hence, in spite of a robust antibody response to conserved segments of VlsE, infection-induced sequence changes within the variable regions alter the antigenicity of VlsE. These results provide the first direct evidence of antigenic variation in the VlsE protein.

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 Detection and quantification of Mycobacterium tuberculosis antigen CFP10 in serum and urine for the rapid diagnosis of active tuberculosis disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marva Seifert ◽  
Eva Vargas ◽  
Victor Ruiz-Valdepeñas Montiel ◽  
Joseph Wang ◽  
Timothy C. Rodwell ◽  
...  
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Specific Antigen ◽  
Active Tuberculosis ◽  
Serum Samples ◽  
Tuberculosis Patients ◽  
Tuberculosis Antigen ◽  
Highly Sensitive ◽  
Tuberculosis Disease ◽  
Serum And Urine

AbstractOutside of the ongoing COVID-19 pandemic, tuberculosis is the leading cause of infectious disease mortality globally. Currently, there is no commercially available point-of-care diagnostic that is rapid, inexpensive, and highly sensitive for the diagnosis of active tuberculosis disease. Here we describe the development and optimization of a novel, highly sensitive prototype bioelectronic tuberculosis antigen (BETA) assay to detect tuberculosis-specific antigen, CFP10, in small-volume serum and urine samples. In this proof-of-concept study we evaluated the performance of the BETA assay using clinical specimens collected from presumptive tuberculosis patients from three independent cohorts. Circulating CFP10 antigen was detected in ALL serum (n = 19) and urine (n = 3) samples from bacteriologically confirmed tuberculosis patients who were untreated or had less than one week of treatment at time of serum collection, successfully identifying all culture positive tuberculosis patients. No CFP10 antigen was detected in serum (n = 7) or urine (n = 6) samples from individuals who were determined to be negative for tuberculosis disease. Additionally, antigen quantification using the BETA assay of paired serum samples collected from tuberculosis patients (n = 8) both before and after treatment initiation, indicate consistently declining within-person levels of CFP10 antigen during treatment. This novel, low-cost assay demonstrates potential as a rapid, non-sputum-based, point-of-care tool for the diagnosis of tuberculosis disease.

scholarly journals A Sensitive Aptamer-Based Biosensor for Electrochemical Quantification of PSA as a Specific Diagnostic Marker of Prostate Cancer

2020 ◽  
Vol 23 ◽  
pp. 243-258 ◽  
Author(s):  
Shokoufeh Hassani ◽  
Armin Salek Maghsoudi ◽  
Milad Rezaei Akmal ◽  
Soheila Rahmani Rahmani ◽  
Pouria Sarihi ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Electrochemical Impedance ◽  
Limit Of Detection ◽  
Specific Antigen ◽  
Differential Pulse ◽  
Label Free ◽  
Serum Samples ◽  
Highly Sensitive ◽  
Set Up

Purpose: The current project aimed to design a simple, highly sensitive, and economical label-free electrochemical aptasensor for determination of prostate-specific antigen (PSA), as the gold standard biomarker for prostate cancer diagnosis. The aptasensor was set up using a screen-printed carbon electrode (SPCE) modified by gold nanoparticles (Au NPs) conjugated to thiolated aptamers. Methods: Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were implemented for electrochemical (EC) characterization of the aptasensor. The determination of PSA was also performed through differential pulse voltammetry (DPV) in [Fe (CN) 6]3-/4- electrolyte solution. Results: The present aptasensor was shown an outstanding linear response in the concentration range of 1 pg/mL - 200 ng/mL with a remarkably lower limit of detection of 0.077 pg/mL. The optimum concentration for PSA separation and the optimum incubation time for antigen-aptamer binding were determined by observing and electing the highest electrochemical responses in a specified time or concentration. Conclusion: According to the results of the specificity tests, the designed aptasensor did not show any significant interactions with other analytes in real samples. Clinical functionality of the aptasensor was appraised in serum samples of healthy individuals and patients examining the PSA level through the fabricated aptasensor and the reference methods. Both methods are comparable in sensitivity. The present fabricated PSA aptasensor with substantial characteristics of ultra-sensitivity and cost-effectiveness can be conventionally built and used for the routine check-up of the men for prostate problems.

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

2014 ◽  
Vol 21 (8) ◽  
pp. 1094-1105 ◽  
Author(s):  
Micah D. Halpern ◽  
Claudia R. Molins ◽  
Martin Schriefer ◽  
Mollie W. Jewett
Keyword(s):  
Lyme Disease ◽  
Effective Means ◽  
Disease Patient ◽  
Healthy Population ◽  
Igg Antibodies ◽  
Serum Samples ◽  
Content Type ◽  
Laboratory Confirmation ◽  
Coefficients Of Variation ◽  
Objective Detection

ABSTRACTA serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detectingBorrelia burgdorferiantibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n= 36) were analyzed. Both of these parameters were found to have coefficients of variation of <3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n= 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n= 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n= 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies againstB. burgdorferi.

scholarly journals Surveillance of Ticks and Tick-Borne Pathogens in Suburban Natural Habitats of Central Maryland

2021 ◽  
Author(s):  
Matthew T Milholland ◽  
Lars Eisen ◽  
Robyn M Nadolny ◽  
Andrias Hojgaard ◽  
Erika T Machtinger ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Spotted Fever ◽  
Babesia Microti ◽  
Borrelia Burgdorferi Sensu Lato ◽  
Spotted Fever Group ◽  
Borrelia Miyamotoi ◽  
Natural Habitats ◽  
Questing Ticks ◽  
Tick Vectors

Abstract Lyme and other tick-borne diseases are increasing in the eastern United States and there is a lack of research on integrated strategies to control tick vectors. Here we present results of a study on tick-borne pathogens detected from tick vectors and rodent reservoirs from an ongoing 5-yr tick suppression study in the Lyme disease-endemic state of Maryland, where human-biting tick species, including Ixodes scapularis Say (Acari: Ixodidae) (the primary vector of Lyme disease spirochetes), are abundant. During the 2017 tick season, we collected 207 questing ticks and 602 ticks recovered from 327 mice (Peromyscus spp. (Rodentia: Cricetidae)), together with blood and ear tissue from the mice, at seven suburban parks in Howard County. Ticks were selectively tested for the presence of the causative agents of Lyme disease (Borrelia burgdorferi sensu lato [s.l.]), anaplasmosis (Anaplasma phagocytophilum), babesiosis (Babesia microti), ehrlichiosis (Ehrlichia ewingii, Ehrlichia chaffeensis, and ‘Panola Mountain’ Ehrlichia) and spotted fever group rickettsiosis (Rickettsia spp.). Peromyscus ear tissue and blood samples were tested for Bo. burgdorferi sensu stricto (s.s), A. phagocytophilum, Ba. microti, and Borrelia miyamotoi. We found 13.6% (15/110) of questing I. scapularis nymphs to be Bo. burgdorferi s.l. positive and 1.8% (2/110) were A. phagocytophilum positive among all sites. Borrelia burgdorferi s.s. was found in 71.1% (54/76) of I. scapularis nymphs removed from mice and 58.8% (194/330) of captured mice. Results from study on tick abundance and pathogen infection status in questing ticks, rodent reservoirs, and ticks feeding on Peromyscus spp. will aid efficacy evaluation of the integrated tick management measures being implemented.

scholarly journals Development of Sensitive Immunoassays for Free and Total Human Glandular Kallikrein 2

2004 ◽  
Vol 50 (9) ◽  
pp. 1607-1617 ◽  
Author(s):  
Ville Väisänen ◽  
Susann Eriksson ◽  
Kaisa K Ivaska ◽  
Hans Lilja ◽  
Martti Nurmi ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Detection Limit ◽  
Solid Phase ◽  
Specific Antigen ◽  
Control Group ◽  
Serum Samples ◽  
Human Kallikrein 2 ◽  
Kallikrein 2 ◽  
Capture Antibody ◽  
Total Psa

Abstract Background: Free and total human kallikrein 2 (hK2) might improve the discrimination between prostate cancer and benign prostatic hyperplasia. Concentrations of hK2 are 100-fold lower than concentrations of prostate-specific antigen (PSA); therefore, an hK2 assay must have a low detection limit and good specificity. Methods: PSA- and hK2-specific monoclonal antibodies were used in solid-phase, two-site immunofluorometric assays to detect free and total hK2. The total hK2 assay used PSA-specific antibodies to block nonspecific signal. The capture antibody of the free hK2 assay did not cross-react with PSA. To determine the hK2 concentrations in the male bloodstream, total hK2 was measured in a control group consisting of 426 noncharacterized serum samples. Free and total hK2 were measured in plasma from 103 patients with confirmed prostate cancer. Results: All 426 males in the control group had a total hK2 concentration above the detection limit of 0.0008 μg/L. The median total hK2 concentration was 0.022 μg/L (range, 0.0015–0.37 μg/L). hK2 concentrations were 0.1–58% of total PSA (median, 3.6%). hK2 concentrations were similar in men 41–50 and 51–60 years of age. The ratio of hK2 to PSA steadily decreased from 5–30% at PSA &lt;1 μg/L to 1–2% at higher PSA concentrations. In 103 patients with prostate cancer, the median hK2 concentration in plasma was 0.079 μg/L (range, 0.0015–16.2 μg/L). The median free hK2 concentration was 0.070 (range, 0.005–12.2) μg/L. The proportion of free to total hK2 varied from 17% to 131% (mean, 85%). Conclusions: The wide variation in the free-to-total hK2 ratio suggests that hK2 in blood plasma is not consistently in the free, noncomplexed form in patients with prostate cancer. The new assay is sufficiently sensitive to be used to study the diagnostic accuracies of free and total hK2 for prostate cancer.

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