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

ABSTRACTThe 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 fromBorrelia 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 fiveB. burgdorferiantigens, consisting of OspC, OspA, DbpA, OppA2, and the C6 peptide, were combined into a fluorescent cytometric bead-based assay for the detection ofB. burgdorferiantigen-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.

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

Clinical and Vaccine Immunology ◽

10.1128/cvi.00608-12 ◽

2013 ◽

Vol 20 (4) ◽

pp. 474-481 ◽

Cited By ~ 27

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.

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Detection of Immune Complexes Is Not Independent of Detection of Antibodies in Lyme Disease Patients and Does Not Confirm Active Infection with Borrelia burgdorferi

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.12.9.1036-1040.2005 ◽

2005 ◽

Vol 12 (9) ◽

pp. 1036-1040 ◽

Cited By ~ 14

Author(s):

Adriana R. Marques ◽

Ronald L. Hornung ◽

Len Dally ◽

Mario T. Philipp

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Rhesus Macaques ◽

Protein A ◽

Surface Protein ◽

Laboratory Diagnosis ◽

Enzyme Linked Immunosorbent Assay ◽

Active Infection ◽

Serum Samples ◽

Outer Surface Protein A

ABSTRACT The Borrelia burgdorferi-specific immune complex (IC) test, which uses polyethylene glycol (PEG) precipitation to isolate ICs from serum, has been used as a research test in the laboratory diagnosis of early Lyme disease (LD) and has been proposed as a marker of active infection. We examined whether B. burgdorferi-specific antibodies were present within PEG-precipitated ICs (PEG-ICs) in patients with LD, posttreatment Lyme disease syndrome, and controls, including individuals who received the outer surface protein A (OspA) vaccine. Using a B. burgdorferi whole-cell enzyme-linked immunosorbent assay (ELISA), we obtained positive PEG-IC results not only in patients with a history of LD, but also in individuals vaccinated with OspA vaccine. The frequency of positive PEG-IC ELISAs in OspA vaccinees was significantly higher with ELISA-reactive than with ELISA-negative unprocessed serum samples (P = 0.001), demonstrating dependency between the tests. Similar results were found using samples from rhesus macaques infected with B. burgdorferi, uninfected macaques vaccinated with OspA, and controls. Therefore, testing for the presence of antibodies against B. burgdorferi in PEG-IC preparations is not more likely to reflect active infection than testing in unprocessed serum and should not be used in individuals who received the OspA vaccine.

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Use of a Toxin Neutralization Assay To Characterize the Serologic Response to Adenylate Cyclase Toxin after Infection with Bordetella pertussis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00370-16 ◽

2016 ◽

Vol 24 (1) ◽

Cited By ~ 4

Author(s):

Joshua C. Eby ◽

Mary C. Gray ◽

Jason M. Warfel ◽

Tod J. Merkel ◽

Erik L. Hewlett

Keyword(s):

Adenylate Cyclase ◽

Bordetella Pertussis ◽

Neutralization Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Immunologic Response ◽

Serum Samples ◽

Content Type ◽

Adenylate Cyclase Toxin ◽

Strong Candidate ◽

Candidate Antigen

ABSTRACT Adenylate cyclase toxin (ACT) is an essential virulence factor of Bordetella pertussis, and antibodies to ACT protect against B. pertussis infection in mice. The toxin is therefore a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG enzyme-linked immunosorbent assay (ELISA) results neutralized ACT cytotoxicity. The toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by PCR, anti-ACT IgG ELISA was positive in 72%, and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA, and none of the samples from humans without pertussis neutralized toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination.

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C-Terminal Invariable Domain of VlsE Is Immunodominant but Its Antigenicity Is Scarcely Conserved among Strains of Lyme Disease Spirochetes

Infection and Immunity ◽

10.1128/iai.69.5.3224-3231.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 3224-3231 ◽

Cited By ~ 17

Author(s):

Fang Ting Liang ◽

Lisa C. Bowers ◽

Mario T. Philipp

Keyword(s):

Lyme Disease ◽

Antibody Response ◽

Human Serum ◽

Synthetic Peptide ◽

Species Complex ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Variable Surface ◽

Entire Sequence ◽

Carboxyl Terminal

ABSTRACT VlsE, the variable surface antigen of Borrelia burgdorferi, contains two invariable domains located at the amino and carboxyl terminal ends, respectively, and a central variable domain. In this study, both immunogenicity and antigenic conservation of the C-terminal invariable domain were assessed. Mouse antiserum to a 51-mer synthetic peptide (Ct) which reproduced the entire sequence of the C-terminal invariable domain of VlsE from B. burgdorferi strain B31 was reacted on immunoblots with whole-cell lysates extracted from spirochetes of 12 strains from the B. burgdorferi sensu lato species complex. The antiserum recognized only VlsE from strain B31, indicating that epitopes of this domain differed among these strains. When Ct was used as enzyme-linked immunosorbent assay (ELISA) antigen, all of the seven monkeys and six mice that were infected with B31 spirochetes produced a strong antibody response to this peptide, indicating that the C-terminal invariable domain is immunodominant. None of 12 monkeys and only 11 of 26 mice that were infected with strains other than B31 produced a detectable anti-Ct response, indicating a limited antigenic conservation of this domain among these strains. Twenty-six of 33 dogs that were experimentally infected by tick inoculation were positive by the Ct ELISA, while only 5 of 18 serum samples from dogs clinically diagnosed with Lyme disease contained detectable anti-Ct antibody. Fifty-seven of 64 serum specimens that were collected from American patients with Lyme disease were positive by the Ct ELISA, while only 12 of 21 European samples contained detectable anti-Ct antibody. In contrast, antibody to the more conserved invariable region IR6 of VlsE was present in all of these dog and human serum samples.

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Diagnosis of alveolar echinococcosis using immunoblotting with plural low molecular weight antigens

Journal of Helminthology ◽

10.1017/s0022149x08116510 ◽

2009 ◽

Vol 83 (1) ◽

pp. 57-61 ◽

Cited By ~ 7

Author(s):

K. Yamano ◽

A. Goto ◽

M. Miyoshi ◽

K. Furuya ◽

Y. Sawada ◽

...

Keyword(s):

Molecular Weight ◽

Alveolar Echinococcosis ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Serological Screening ◽

Screening Process ◽

Medical Practitioners ◽

Specific Reactions ◽

Hospital Outpatients ◽

Higher Sensitivity

AbstractAlveolar echinococcosis (AE) is endemic to Hokkaido, Japan. For the past 20 years, detection of AE among inhabitants has involved serological screening using an enzyme-linked immunosorbent assay (ELISA) followed by Western blotting (WB). Between the years 1987 and 2000, antigens targeted on 66, 55 and 30–35 kDa bands were routinely used in the WB step of AE diagnosis. However, since 2001 diagnosis has been dependent on three smaller molecular weight antigens (26–28, 18 and 7–8 kDa). Due to its higher sensitivity, this improved WB approach has been used as a confirmation step in the screening process and also for the testing of suspected AE cases in hospital outpatients. Using the improved WB technique, a total of 1745 serum samples were examined in 2001–2006 with 81 patients detected and registered with AE. Interestingly, sera from 76 of the 81 diagnosed AE patients (93.8%) demonstrated reactivity with all three antigens. However, sera from the remaining five patients (6.2%) demonstrated no reactivity with the 18 kDa antigen, even though they exhibited clearly detectable levels of reactivity with the 26–28 and 7–8 kDa bands. These results suggest that medical practitioners need to pay particular attention to the specific reactions to some different diagnostic antigens to minimize the risk of misdiagnosing AE patients. In turn, these results may also provide important diagnostic information for cystic echinococcosis (CE).

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Quantitative Approach for the Serodiagnosis of Canine Lyme Disease by the Immunoblot Procedure

Journal of Clinical Microbiology ◽

10.1128/jcm.38.7.2628-2632.2000 ◽

2000 ◽

Vol 38 (7) ◽

pp. 2628-2632 ◽

Cited By ~ 11

Author(s):

Marta A. Guerra ◽

Edward D. Walker ◽

Uriel Kitron

Keyword(s):

Logistic Regression ◽

Lyme Disease ◽

Natural Infection ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Immunoblot Assay ◽

Serological Status ◽

Low Probability ◽

Positive Results ◽

Logistic Regression Equation

Serum samples obtained from healthy, asymptomatic dogs in areas of Wisconsin and northern Illinois where Lyme disease is endemic or nonendemic were assayed for antibodies to Borrelia burgdorferi by enzyme-linked immunosorbent assay (ELISA), and positive results were confirmed by immunoblot assay. We found that 56.9% (562 of 1,077) of the samples were positive by ELISA and 82.0% (461 of 562) were positive by immunoblotting. A logistic regression model was developed to distinguish between nonvaccinated dogs naturally infected with B. burgdorferi from areas where the disease is endemic and dogs from areas where the disease is nonendemic that were vaccinated against Lyme disease. Of the 18 protein bands analyzed, 8 were found to be significantly different (P < 0.05) between the two groups. p93, p34, p31, and p28 occurred with increased frequency in vaccinated dogs, while p58, p37, p35, and p30 occurred more frequently in naturally infected dogs. The logistic regression equation obtained was used to determine the probability of natural infection among vaccinated dogs residing in areas where the disease is endemic. Of 125 samples, 87.2% had a very low probability of natural infection and only 2.4% were highly likely to be infected. Logistic regression is a useful method for distinguishing between vaccinated and naturally infected dogs and predicting the serological status of vaccinated dogs from areas where Lyme disease is endemic.

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Immunogenicity of heterologous prime/booster-inactivated and adenoviral-vectored COVID-19 vaccine: real-world data

10.21203/rs.3.rs-785693/v1 ◽

2021 ◽

Author(s):

Nasamon Wanlapakorn ◽

Nungruthai Suntronwong ◽

Harit Phowatthanasathian ◽

Ritthideach Yorsang ◽

Thanunrat Thongmee ◽

...

Keyword(s):

Neutralizing Antibody ◽

Enzyme Linked Immunosorbent Assay ◽

Antibody Activity ◽

Serum Samples ◽

Real World Data ◽

Comparison Groups ◽

Chemiluminescent Microparticle Immunoassay ◽

Specific Igg ◽

Vectored Vaccine ◽

Sera Samples

Abstract Limited data are available on the responses to heterologous vaccine regimens for SARS-CoV-2, especially among countries using inactivated and adenoviral-vectored vaccines. A total of 77 participants who received heterologous prime/booster-inactivated COVID-19 vaccine and adenoviral-vectored vaccine were enrolled in our study. There were two comparison groups vaccinated with the homologous CoronaVac (N = 79) and AZD1222 (N = 80) regimen. All sera samples were tested for SARS-CoV-2 spike receptor-binding-domain (RBD) IgG using a chemiluminescent microparticle immunoassay (CMIA). The neutralizing activity in a subset of serum samples was tested against the original Wuhan strain and variants of concern, B.1.1.7 and B.1.351, using an enzyme-linked immunosorbent assay (ELISA)-based surrogate virus neutralization test (sVNT). The CoronaVac followed by the AZD1222 vaccine induced higher levels of spike RBD-specific IgG than that of two-dose CoronaVac or AZD1222 vaccines (p < 0.001). Sera samples of the CoronaVac/AZD1222 vaccine recipients elicited higher neutralizing antibody activity against the original Wuhan and the B.1.351 strain than in the recipients of the two-dose CoronaVac or AZD1222. Following the inactivated CoronaVac/adenoviral-vectored (AZD1222) vaccination administered 14–72 days apart, participants receiving the heterologous vaccine regimen had higher spike RBD-specific IgG and neutralizing activities than the homologous CoronaVac vaccine recipients.

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Mosquito Bite-Induced Controlled Human Malaria Infection withPlasmodium vivaxorP. falciparumGenerates Immune Responses to Homologous and Heterologous Preerythrocytic and Erythrocytic Antigens

Infection and Immunity ◽

10.1128/iai.00541-18 ◽

2018 ◽

Vol 87 (3) ◽

Cited By ~ 7

Author(s):

Cysha E. Hall ◽

Lisa M. Hagan ◽

Elke Bergmann-Leitner ◽

Donna M. Tosh ◽

Jason W. Bennett ◽

...

Keyword(s):

Malaria Infection ◽

Surface Protein ◽

Merozoite Surface Protein ◽

Similar Proportion ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Content Type ◽

Human Malaria ◽

Before And After ◽

Controlled Human Malaria Infection

ABSTRACTSeroepidemiological studies on the prevalence of antibodies to malaria antigens are primarily conducted on individuals from regions of endemicity. It is therefore difficult to accurately correlate the antibody responses to the timing and number of prior malaria infections. This study was undertaken to assess the evolution of antibodies to the dominant surface antigens ofPlasmodium vivaxandP. falciparumfollowing controlled human malaria infection (CHMI) in malaria-naive individuals. Serum samples from malaria-naive adults, collected before and after CHMI with eitherP. vivax(n= 18) orP. falciparum(n= 18), were tested for the presence of antibodies to the circumsporozoite protein (CSP) and the 42-kDa fragment of merozoite surface protein 1 (MSP-142) ofP. vivaxandP. falciparumusing an enzyme-linked immunosorbent assay (ELISA). Approximately 1 month following CHMI with eitherP. vivaxorP. falciparum, >60% of subjects seroconverted to homologous CSP and MSP-1. More than 50% of the subjects demonstrated reactivity to heterologous CSP and MSP-142, and a similar proportion of subjects remained seropositive to homologous MSP-142>5 months after CHMI. Computational analysis provides insight into the presence of cross-reactive responses. The presence of long-lived and heterologous reactivity and its functional significance, if any, need to be taken into account while evaluating malaria exposure in field settings.

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SOX Antibodies in Small-Cell Lung Cancer and Lambert-Eaton Myasthenic Syndrome: Frequency and Relation With Survival

Journal of Clinical Oncology ◽

10.1200/jco.2008.20.6169 ◽

2009 ◽

Vol 27 (26) ◽

pp. 4260-4267 ◽

Cited By ~ 100

Author(s):

Maarten J. Titulaer ◽

Rinse Klooster ◽

Marko Potman ◽

Lidia Sabater ◽

Francesc Graus ◽

...

Keyword(s):

Western Blot ◽

High Throughput Screening ◽

Small Cell Lung Carcinoma ◽

Diagnostic Value ◽

Small Cell ◽

Enzyme Linked Immunosorbent Assay ◽

Small Cell Lung ◽

Serum Samples ◽

Cell Lung Carcinoma ◽

Myasthenic Syndrome

Purpose SOX1 antibodies are common in small-cell lung carcinoma (SCLC) with and without paraneoplastic syndrome (PNS) and can serve as serological tumor marker. Addition of other antibodies might improve its diagnostic power. We validated an enzyme-linked immunosorbent assay (ELISA) to assess the diagnostic value of serum antibodies in SCLC and Lambert-Eaton myasthenic syndrome (LEMS). Clinical outcome with respect to SOX antibodies was evaluated, as the SOX-related antitumor immune response might help to control the tumor growth. Patients and Methods We used recombinant SOX1, SOX2, SOX3, SOX21, HuC, HuD, or HelN1 proteins in an ELISA to titrate serum samples and validated the assay by western blot. We tested 136 consecutive SCLC patients, 86 LEMS patients (43 with SCLC), 14 patients with SCLC and PNS (paraneoplastic cerebellar degeneration or Hu syndrome), 62 polyneuropathy patients, and 18 healthy controls. Results Our ELISA was equally reliable as western blot. Forty-three percent of SCLC patients and 67% of SCLC-LEMS patients had antibodies to one of the SOX or Hu proteins. SOX antibodies had a sensitivity of 67% and a specificity of 95% to discriminate between LEMS with SCLC and nontumor LEMS. No difference in survival was observed between SOX positive and SOX negative SCLC patients. Conclusion SOX antibodies are specific serological markers for SCLC. Our assay is suitable for high throughput screening, detecting 43% of SCLC. SOX antibodies have diagnostic value in discriminating SCLC-LEMS from nontumor LEMS, but have no relation to survival in patients with SCLC.

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Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

Clinical and Vaccine Immunology ◽

10.1128/cvi.00583-13 ◽

2013 ◽

Vol 21 (1) ◽

pp. 96-106 ◽

Cited By ~ 18

Author(s):

Lourena E. Costa ◽

Mayara I. S. Lima ◽

Miguel A. Chávez-Fumagalli ◽

Daniel Menezes-Souza ◽

Vivian T. Martins ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Phage Display ◽

Leishmania Infantum ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Phage Display Technology ◽

Predictive Values ◽

Content Type ◽

Phage Elisa

ABSTRACTVisceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy andTrypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected withLeishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n= 31) compared to those from vaccinated dogs (n= 21), experimentally infected dogs with cross-reactive parasites (n= 23), and healthy controls (n= 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no cross-reactivity withT. cruzi- orEhrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes ofL. infantumantigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs.

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