Development of a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycoplasma bovis Infection in Cattle

ABSTRACTMycoplasma boviscauses a range of diseases in cattle, including mastitis, arthritis, and pneumonia. However, accurate serological diagnosis of infection remains problematic. The studies described here aimed to identify an antigen that might be used to develop a more specific and sensitive diagnostic assay. A 226-kDa immunogenic protein was consistently detected in Western blots by antibodies in sera from calves experimentally infected withM. bovis. This protein was shown to be a membrane protein with lipase activity and was named mycoplasma immunogenic lipase A (MilA). Different regions of MilA were expressed inEscherichia colias glutathioneS-transferase (GST) fusion proteins and recombinant products from the amino-terminal end shown to have strong immunoreactivity withM. bovis-specific bovine sera. The most immunoreactive fusion protein, GST-MilA-ab, was used to develop indirect IgM and IgG enzyme-linked immunosorbent assays (ELISAs). The IgM ELISA detectedM. bovis-specific IgM antibody 2 weeks after infection with 97.1% sensitivity and had a specificity of 63.3%, while the IgG ELISA detectedM. bovis-specific IgG 3 weeks after infection with 92.86% sensitivity and had a specificity of 98.7%, demonstrating that the IgG ELISA has potential for use as a sensitive and specific assay for detecting infection in cattle.

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Comparison of Western Immunobloting to an Enzyme-Linked Immunosorbent Assay for the Determination of Anti-Bordetella pertussis Antibodies

Clinical and Vaccine Immunology ◽

10.1128/cvi.00450-10 ◽

2011 ◽

Vol 18 (4) ◽

pp. 615-620 ◽

Cited By ~ 1

Author(s):

Stephen D. Merrigan ◽

Ryan J. Welch ◽

Christine M. Litwin

Keyword(s):

Immunosorbent Assay ◽

Bordetella Pertussis ◽

World Health ◽

Enzyme Linked Immunosorbent Assay ◽

Recent Infection ◽

Content Type ◽

Igm Elisa ◽

Iga Antibodies ◽

Health Organization

ABSTRACTDuringBordetella pertussisinfection, it has been established that an increase of anti-pertussis toxin (PT) and anti-filamentous hemagglutinin (FHA) antibodies occurs. Immunoblots from two manufacturers using FHA and PT antigens were compared with an enzyme-linked immunosorbent assay (ELISA) that used both FHA and PT. One manufacturer used two concentrations of PT bands for the IgG immunoblot, calibrated to the World Health Organization standard for PT in international units (IU/ml), 100 IU/ml (PT-100) and 8 IU/ml (PT). The second immunoblot kit measured antibodies to a single calibrated PT band. Both kits measured IgA antibodies, and one additionally measured IgM antibodies. Two of 41 (5%) ELISA IgM positives were confirmed positive by IgM immunoblotting, suggesting poor specificity of the IgM ELISA. The agreements of the IgG and IgA immunoblots with the ELISA ranged from 72.5% to 85.3%, with only 38 to 51% of IgA positives confirmed by immunoblotting and only 61 to 68% of IgG positives confirmed by immunoblotting. The two immunoblots correlated well with each other, with 91.7% and 94.3% agreement for IgG and IgA, respectively. When the FHA band was used with the PT band as the criterion for positivity, significant differences existed in specificity compared to the ELISA (IgG, 84.1% versus 33.3%; IgA, 82.4% versus 71.0%). When the positive IgA immunoblots (evidence of natural recent infection) were compared to the positive PT-100 IgG immunoblots (evidence of recent infection or vaccination), the PT-100 blot showed a 71% sensitivity in detecting natural recent infection.B. pertussisimmunoblots, alone or in combination with ELISAs, can aid in the diagnosis ofB. pertussisinfection.

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Improved Serodiagnostic Performance for Lyme Disease by Use of Two Recombinant Proteins in Enzyme-Linked Immunosorbent Assay Compared to Standardized Two-Tier Testing

Journal of Clinical Microbiology ◽

10.1128/jcm.01004-17 ◽

2017 ◽

Vol 55 (10) ◽

pp. 3046-3056 ◽

Cited By ~ 4

Author(s):

Gary L. Bradshaw ◽

R. Kelley Thueson ◽

Todd J. Uriona

Keyword(s):

Lyme Disease ◽

Recombinant Proteins ◽

Immunosorbent Assay ◽

Negative Control ◽

Full Length ◽

Test Method ◽

Stage I ◽

Enzyme Linked Immunosorbent Assay ◽

Western Blots ◽

Content Type

ABSTRACTThe most reliable test method for the serological confirmation of Lyme disease (LD) is a 2-tier method recommended by the CDC in 1995. The first-tier test is a low-specificity enzyme-linked immunosorbent assay (ELISA), and the second-tier tests are higher-specificity IgG and IgM Western blots. This study describes the selection of twoBorrelia burgdorferirecombinant proteins and evaluation of their performance in a simple 1-tier test for the serological confirmation of LD. These two proteins were generated from (i) the full-lengthdbpAgene combined with the invariable region 6 of thevlsEgene (DbpA/C6) and (b) the full-lengthospCgene (OspC). The expressed DbpA/C6 and OspC proteins were useful in detecting anti-BorreliaIgG and IgM antibodies, respectively. A blind study was conducted on a well-characterized panel of 279 human sera from the CDC, comparing ELISAs using these two recombinant antigens with the 2-tier test method. The two methods (DbpA/C6-OspC versus 2-tier test) were equivalent in identifying sera from negative-control subjects (99% and 100% specificity, respectively) and in detecting stage II and III LD patient sera (100% and 100% sensitivity). However, the DbpA/C6-OspC ELISA was markedly better (80% versus 63%) than the 2-tier test method in detecting anti-Borreliaantibodies in stage I LD patients. The findings suggest that these antigens could be used in a simple 1-tier ELISA that is faster to perform, easier to interpret, and less expensive than the 2-tier test method and which is better at detectingBorrelia-specific antibodies in sera from patients with stage I LD.

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Characterization of Immunodominant Linear B-Cell Epitopes on the Carboxy Terminus of the Rinderpest Virus Nucleocapsid Protein

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.4.658-664.2004 ◽

2004 ◽

Vol 11 (4) ◽

pp. 658-664 ◽

Cited By ~ 11

Author(s):

Kang-Seuk Choi ◽

Jin-Ju Nah ◽

Young-Joon Ko ◽

Shien-Young Kang ◽

Kyoung-Jin Yoon ◽

...

Keyword(s):

Antigenic Site ◽

Enzyme Linked Immunosorbent Assay ◽

Carboxy Terminus ◽

Rinderpest Virus ◽

N Protein ◽

Amino Terminal ◽

Immunogenic Epitope ◽

Carboxy Terminal ◽

Gst Fusion Proteins

ABSTRACT The nucleocapsid (N) protein of rinderpest virus (RPV) is one of the most abundant and immunogenic viral proteins expressed during natural or experimental infection. To identify immunogenic epitopes on the N protein, different forms of RPV N protein, including the full-length protein (N1-525), an amino-terminal construct (N1-179), and a carboxy-terminal construct (N414-496), were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The antigenicity of each recombinant protein was evaluated by Western immunoblotting. All recombinants were recognized by hyperimmune RPV bovine antisera, indicating that immunoreactive epitopes may be present at both ends of the N protein. However, GST-N414-496 was much more antigenic than GST-N1-179 when tested with sera from vaccinated cattle, suggesting that an immunodominant or highly immunogenic epitope(s) may be located at the carboxy terminus of the N protein. Epitope mapping with overlapping peptides representing different regions of the carboxy terminus (amino acids 415 to 524) revealed three nonoverlapping antigenic sites in regions containing the residues 440VPQVRKETRASSR452 (site 1), 479PEADTDPL486 (site 2), and 520DKDLL524 (site 3). Among these, antigenic site 2 showed the strongest reactivity with hyperimmune anti-RPV bovine sera in a peptide enzyme-linked immunosorbent assay but did not react with hyperimmune caprine sera raised against peste-des-petits-ruminants virus, which is antigenically closely related to RPV. Identification of an immunodominant linear antigenic site at the carboxy terminus of the N protein may provide an antigen basis for designing diagnostics specific for RPV.

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In Vivo-Expressed Proteins of Virulent Leptospira interrogans Serovar Autumnalis N2 Elicit Strong IgM Responses of Value in Conclusive Diagnosis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00509-15 ◽

2015 ◽

Vol 23 (1) ◽

pp. 65-72 ◽

Cited By ~ 8

Author(s):

Veerapandian Raja ◽

Santhanam Shanmughapriya ◽

Murugesan Kanagavel ◽

Sergey C. Artiushin ◽

Sridhar Velineni ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Recombinant Proteins ◽

Natural Infection ◽

Enzyme Linked Immunosorbent Assay ◽

Leptospira Interrogans ◽

Content Type ◽

Igm Elisa ◽

Conclusive Diagnosis

ABSTRACTLeptospirosis is a serious zoonosis that is underdiagnosed because of limited access to laboratory facilities in Southeast Asia, Central and South America, and Oceania. Timely diagnosis of locally distributed serovars of high virulence is crucial for successful care and outbreak management. Using pooled patient sera, an expression gene library of a virulentLeptospira interrogansserovar Autumnalis strain N2 isolated in South India was screened. The identified genes were characterized, and the purified recombinant proteins were used as antigens in IgM enzyme-linked immunosorbent assay (ELISA) either singly or in combination. Sera (n= 118) from cases of acute leptospirosis along with sera (n= 58) from healthy subjects were tested for reactivity with the identified proteins in an ELISA designed to detect specific IgM responses. We have identified nine immunoreactive proteins, ArgC, RecA, GlpF, FliD, TrmD, RplS, RnhB, Lp28.6, and Lrr44.9, which were found to be highly conserved among pathogenic leptospires. Apparently, the proteins ArgC, RecA, GlpF, FliD, TrmD, and Lrr44.9 are expressed during natural infection of the host and undetectable inin vitrocultures. Among all the recombinant proteins used as antigens in IgM ELISA, ArgC had the highest sensitivity and specificity, 89.8% and 95.5%, respectively, for the conclusive diagnosis of leptospirosis. The use of ArgC and RecA in combination for IgM ELISA increased the sensitivity and specificity to 95.7% and 94.9%, respectively. ArgC and RecA thus elicited specific IgM responses and were therefore effective in laboratory confirmation ofLeptospirainfection.

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Evaluation of Enzyme-Linked Immunosorbent Assays for Detection of Mycoplasma bovis-Specific Antibody in Bison Sera

Clinical and Vaccine Immunology ◽

10.1128/cvi.00409-13 ◽

2013 ◽

Vol 20 (9) ◽

pp. 1405-1409 ◽

Cited By ~ 8

Author(s):

Karen B. Register ◽

Randy E. Sacco ◽

Steven C. Olsen

Keyword(s):

Infectious Disease ◽

Protein G ◽

Mycoplasma Bovis ◽

Content Type ◽

Specific Serum ◽

History Of ◽

Specific Igg ◽

Immunosorbent Assays ◽

Comparison Of The Results ◽

Antibody Levels

ABSTRACTMycoplasma bovishas recently emerged as a significant and costly infectious disease problem in bison. A method for the detection ofM. bovis-specific serum antibodies is needed in order to establish prevalence and transmission patterns. Enzyme-linked immunosorbent assays (ELISAs) validated for the detection ofM. bovis-specific serum IgG in cattle are commercially available, but their suitability for bison sera has not been determined. A collection of bison sera, most from animals with a known history of infection or vaccination withM. bovis, was tested forM. bovis-specific IgG using commercially available kits as well as an in-house ELISA in which either cattle or bisonM. bovisisolates were used as a source of antigen. Comparison of the results demonstrates that ELISAs optimized for cattle sera may not be optimal for the identification of bison seropositive forM. bovis, particularly those with low to moderate antibody levels. The reagent used for the detection of bison IgG and the source of the antigen affect the sensitivity of the assay. Optimal performance was obtained when the capture antigen was derived from bison isolates rather than cattle isolates and when a protein G conjugate rather than an anti-bovine IgG conjugate was used for the detection of bison IgG.

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Assessment of Brucellosis Card test in screening patients for brucellosis

Epidemiology and Infection ◽

10.1017/s0950268800067145 ◽

1988 ◽

Vol 100 (3) ◽

pp. 389-398 ◽

Cited By ~ 4

Author(s):

G. F. Araj ◽

G. M. Brown ◽

M. M. Haj ◽

N. V. Madhvan

Keyword(s):

Elisa Test ◽

Rapid Screening ◽

Enzyme Linked Immunosorbent Assay ◽

Control Groups ◽

Igm Elisa ◽

Microagglutination Test ◽

Rapid Screening Test ◽

Specific Igg ◽

Card Test

SummaryThe Brucellosis Card test (Brewers' Diagnostic Kits, Hynson, Westcott and Dunning, Inc., Baltimore, Md.) was evaluated in relation to the Brucelloslide test (bioMérieux, France), the microagglutination test (MAT) and the demonstration of brucella-specific IgG, IgM and IgA in an enzyme-linked immunosorbent assay (ELISA). A total of 573 serum specimens was tested. These included sera from patients with acute brucellosis (159), chronic brucellosis (23) and patients who had been diagnosed previously as having had brucella infection (155). Control groups consisted of patients with diseases other than brucellosis (52), others with noninfectious diseases (20), and healthy individuals (164). The Card test detected 100% of the patients with acute and 61% of the patients with chronic brucellosis. The sera from the control groups were all negative. Similar results were obtained with the Brucelloslide test and the MAT. The ELISA test detected brucellaspecific Ig of all classes in the serum of patients with acute brucellosis, and IgG and IgA in the serum of patients with chronic brucellosis. In the latter group, IgM was also detected in 32% of the sera. Twenty-three per cent of sera with titres of 20 by the MAT were positive on the Card test and had ELISA titres for IgM, IgG and IgA of 400. Characterization of the antibodies involved in the Card test showed that sera with IgM ELISA titres of 1600, or an IgM titres of 800 together with IgG and IgA titres ≥ 200 were Card test positive. Higher IgG (≥ 1600) plus IgA (≥ 400) titres were required to produce a positive Card test in the absence of IgM or when the IgM titre was ≤ 200. The Card test has a potential value as a rapid screening test for humans with acute brucellosis and shows similar results to Brucelloslide and MAT tests. ELISA, however, remains the most reliable test for diagnosis of brucellosis especially in patients with chronic and complicated stages of the disease.

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Natural and Vaccine-Induced Acquisition of Cross-Reactive IgG-Inhibiting ICAM-1-Specific Binding of a Plasmodium falciparum PfEMP1 Subtype Associated Specifically with Cerebral Malaria

Infection and Immunity ◽

10.1128/iai.00622-17 ◽

2018 ◽

Vol 86 (4) ◽

Cited By ~ 8

Author(s):

Rebecca W. Olsen ◽

Gertrude Ecklu-Mensah ◽

Anja Bengtsson ◽

Michael F. Ofori ◽

John P. A. Lusingu ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Cerebral Malaria ◽

Specific Binding ◽

Intercellular Adhesion Molecule ◽

Enzyme Linked Immunosorbent Assay ◽

Intercellular Adhesion Molecule 1 ◽

Content Type ◽

Protein Receptor ◽

Specific Igg

ABSTRACT Cerebral malaria (CM) is a potentially deadly outcome of Plasmodium falciparum malaria that is precipitated by sequestration of infected erythrocytes (IEs) in the brain. The adhesion of IEs to brain endothelial cells is mediated by a subtype of parasite-encoded erythrocyte membrane protein 1 (PfEMP1) that facilitates dual binding to host intercellular adhesion molecule 1 (ICAM-1) and endothelial protein receptor C (EPCR). The PfEMP1 subtype is characterized by the presence of a particular motif (DBLβ_motif) in the constituent ICAM-1-binding DBLβ domain. The rate of natural acquisition of DBLβ_motif-specific IgG antibodies and the ability to induce such antibodies by vaccination are unknown, and the aim of this study was to provide such data. We used an enzyme-linked immunosorbent assay (ELISA) to measure DBLβ-specific IgG in plasma from Ghanaian children with malaria. The ability of human immune plasma and DBLβ-specific rat antisera to inhibit the interaction between ICAM-1 and DBLβ was assessed using ELISA and in vitro assays of IE adhesion under flow. The acquisition of DBLβ_motif-specific IgG coincided with age-specific susceptibility to CM. Broadly cross-reactive antibodies inhibiting the interaction between ICAM-1 and DBLβ_motif domains were detectable in immune plasma and in sera of rats immunized with specific DBLβ_motif antigens. Importantly, antibodies against the DBLβ_motif inhibited ICAM-1-specific in vitro adhesion of erythrocytes infected by four of five P. falciparum isolates from cerebral malaria patients. We conclude that natural exposure to P. falciparum as well as immunization with specific DBLβ_motif antigens can induce cross-reactive antibodies that inhibit the interaction between ICAM-1 and a broad range of DBLβ_motif domains. These findings raise hope that a vaccine designed specifically to prevent CM is feasible.

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New Commercially Available IgG Kits and Time-Resolved Fluorometric IgE Assay for Diagnosis of Allergic Bronchopulmonary Aspergillosis in Patients with Cystic Fibrosis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00498-15 ◽

2015 ◽

Vol 23 (3) ◽

pp. 196-203 ◽

Cited By ~ 8

Author(s):

Coralie Barrera ◽

Bénédicte Richaud-Thiriez ◽

Steffi Rocchi ◽

Bénédicte Rognon ◽

Sandrine Roussel ◽

...

Keyword(s):

Cystic Fibrosis ◽

Aspergillus Fumigatus ◽

Allergic Bronchopulmonary Aspergillosis ◽

Specific Ige ◽

Enzyme Linked Immunosorbent Assay ◽

Time Resolved ◽

Content Type ◽

Elisa Kit ◽

Fungal Allergens ◽

Specific Igg

ABSTRACTAllergic bronchopulmonary aspergillosis (ABPA) is difficult to diagnose; diagnosis relies on clinical, radiological, pathological, and serological criteria. Our aim was to assess the performance of two new commercially available kits and a new in-house assay: anAspergillus fumigatusenzyme-linked immunosorbent assay (ELISA) IgG kit (Bordier Affinity Products), anAspergillusWestern blotting IgG kit (LDBio Diagnostics), and a new in-house time-resolved fluorometric IgE assay (dissociation-enhanced lanthanide fluorescent immunoassay, or DELFIA) using recombinant proteins from anAspergillussp. recently developed by our laboratory for ABPA diagnosis in a retrospective study that included 26 cystic fibrosis patients.Aspergillus fumigatus-specific IgG levels measured by a commercial ELISA kit were in accordance with the level of precipitins currently used in our lab. The ELISA kit could accelerate and help standardize ABPA diagnosis.Aspergillus fumigatus-specific IgE levels measured by ImmunoCAP (Phadia) withA. fumigatusM3 antigen and by DELFIA with a purified protein extract ofA. fumigatuswere significantly correlated (P< 10−6). The results with recombinant antigens glucose-6-phosphate isomerase and mannitol-1-phosphate dehydrogenase were encouraging but must be confirmed with sera from more patients. The DELFIA is an effective tool that can detect specific IgE against more fungal allergens than can be detected with other commercially available tests.

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

Clinical and Vaccine Immunology ◽

10.1128/cvi.00685-15 ◽

2016 ◽

Vol 23 (4) ◽

pp. 294-303 ◽

Cited By ~ 13

Author(s):

Monica E. Embers ◽

Nicole R. Hasenkampf ◽

Mary B. Barnes ◽

Elizabeth S. Didier ◽

Mario T. Philipp ◽

...

Keyword(s):

Lyme Disease ◽

Rhesus Macaques ◽

Diagnostic Value ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Content Type ◽

Rational Combination ◽

Specific Igg ◽

Sample Set ◽

Higher Sensitivity

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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Utility of Bartonella henselae IgM Western Blot Bands for Serodiagnosis of Cat Scratch Disease

Journal of Clinical Microbiology ◽

10.1128/jcm.01322-17 ◽

2017 ◽

Vol 56 (1) ◽

Cited By ~ 1

Author(s):

Ken-ichiro Otsuyama ◽

Hidehiro Tsuneoka ◽

Hiroka Yoshidomi ◽

Mio Haraguchi ◽

Masashi Yanagihara ◽

...

Keyword(s):

Western Blot ◽

Bartonella Henselae ◽

Immunoglobulin M ◽

Enzyme Linked Immunosorbent Assay ◽

Cat Scratch Disease ◽

Content Type ◽

Specificity And Sensitivity ◽

Igm Elisa ◽

Positive Rate ◽

Healthy Control

ABSTRACTWe evaluated the utility of Western blot (WB) bands ofBartonella henselae in detecting anti-B. henselaeimmunoglobulin M (IgM) for serodiagnosis of cat scratch disease (CSD). IgM band patterns were examined using sera from 92 patients clinically suspected of having CSD and from 130 healthy individuals. Positive WB bands were observed in 49 (53.5%) of the 92 patient sera. Three bands at 8 to 10, 31 to 35, and 70 kDa were regarded as relevant forB. henselaebecause all of the positive sera yielded at least one of the three bands, and none of the healthy control sera showed reactivity to any of them. In contrast, the positive rate of the patient sera by conventional indirect fluorescence antibody assay (IFA) forB. henselaeIgM was 28.3% (26/92) among the patients. These finding suggest that the IgM-WB assay, although cumbersome to perform, can be used for confirmatory diagnosis of CSD with no false positivity in the control sera. Purification of proteins in the specific bands may contribute to the development of an IgM enzyme-linked immunosorbent assay (IgM-ELISA) with improved specificity and sensitivity.

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