Comparative evaluation of the immunodominant proteins of Brucella abortus for the diagnosis of cattle brucellosis

Background and Aim: The present serodiagnosis of brucellosis in livestock is based on the whole cell or smooth lipopolysaccharide of the Brucella organism in which specificity is hampered by the cross-reactivity, especially with the antibodies against Yersinia enterocolitica O:9 organism. The problem can be addressed by screening for better immunodominant antigens. Hence, the present study was undertaken to screen protein antigens of Brucella abortus for their diagnostic potential in cattle brucellosis. Materials and Methods: Protein antigens of B. abortus (n=10) non-reactive to antibodies against Y. enterocolitica O:9 were selected, expressed in Escherichia coli, assessed the reactivity of expressed recombinant proteins by Western blot, standardized indirect-enzyme-linked immunosorbent assay (ELISA) for detecting Brucella antibodies in cattle serum, and comparative evaluation was done. Results: All the selected protein antigens were expressed and in the Western blot with Brucella antibodies positive cattle serum, six recombinant (Brucella protein 26 [BP26], Cu-Zn Superoxide dismutase [SodC], B. abortus I-1885, Serine protease, Bacterioferritin, and Brucella Lumazine Synthase [BLS]) proteins showed reaction whereas none of the proteins showed reactivity with Brucella negative cattle serum. ELISA has been done using known Brucella positive and negative cattle sera samples (n=113 each) in which the performance of recombinant proteins in diagnosing brucellosis was in the order of BP26 > BLS > SodC followed by rest of the proteins. BP26 based ELISA was found to be better with area under the curve as 0.953, and diagnostic sensitivity, diagnostic specificity, and Youden's index of 90.27%, 95.58%, and 0.8584, respectively, with the excellent agreement (k=0.85). Conclusion: BP26 could be a potential diagnostic antigen among the immunodominant proteins of B. abortus in ruling out Y. enterocolitica O:9 infection while diagnosing brucellosis in cattle herds.

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Putative Novel Surface-Exposed Streptococcus agalactiae Protein Frequently Expressed by the Group B Streptococcus from Zimbabwe

Clinical and Vaccine Immunology ◽

10.1128/cvi.00133-09 ◽

2009 ◽

Vol 16 (9) ◽

pp. 1302-1308 ◽

Cited By ~ 6

Author(s):

Rooyen T. Mavenyengwa ◽

Johan A. Maeland ◽

Sylvester R. Moyo

Keyword(s):

Rabbit Antiserum ◽

Periodate Oxidation ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Protein Antigens ◽

Group B Streptococci ◽

Phenotypic Marker ◽

Whole Cell ◽

Strain Collection ◽

Group B

ABSTRACTGroup B streptococci (GBS) express a variety of surface-exposed and strain-variable proteins which function as phenotypic markers and as antigens which are able to induce protective immunity in experimental settings. Among these proteins, the chimeric and immunologically cross-reacting alpha-like proteins are particularly important. Another protein, R3, which has been less well studied, occurred at a frequency of 21.5% in GBS from Zimbabwe and, notably, occurred in serotype V strains at a frequency of 75.9%. Working with rabbit antiserum raised against the R3 reference strain ATCC 49447 (strain 10/84; serotype V/R3) to detect the expression of the R3 protein, we recorded findings which suggested that strain 10/84 expressed a strain-variable protein antigen, in addition to R3. The antigen was detected by various enzyme-linked immunosorbent assay-based tests by using acid extract antigens or GBS whole-cell coats and by whole-cell-based Western blotting. We named the putative novel antigen the Z antigen. The Z antigen was a high-molecular-mass antigen that was susceptible to degradation by pepsin and trypsin but that was resistant tom-periodate oxidation and failed to show immunological cross-reactivity with any of a variety of other GBS protein antigens. The Z antigen was expressed by 33/121 (27.2%) of strains of a Zimbabwean GBS strain collection and by 64.2% and 72.4% of the type Ib and type V strains, respectively, and was occasionally expressed by GBS of other capsular serotypes. Thus, the putative novel GBS protein named Z showed distinct capsular antigen associations and presented as an important phenotypic marker in GBS from Zimbabwe. It may be an important antigen in GBS from larger areas of southern Africa. Its prevalence in GBS from Western countries is not known.

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Combined Use of Indirect ELISA and Western Blotting with Recombinant Hepatocellular Carcinoma-Associated Antigen 59 Is a Potential Immunodiagnostic Tool for the Detection of Prepatent Haemonchus contortus Infection in Goat

Animals ◽

10.3390/ani9080548 ◽

2019 ◽

Vol 9 (8) ◽

pp. 548 ◽

Cited By ~ 7

Author(s):

Muhammad Ali-ul-Husnain Naqvi ◽

Sana Zahra Naqvi ◽

Muhammad Ali Memon ◽

Kalibixiati Aimulajiang ◽

Muhammad Haseeb ◽

...

Keyword(s):

Western Blot ◽

Western Blotting ◽

Haemonchus Contortus ◽

Indirect Elisa ◽

Fasciola Hepatica ◽

False Negative ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Combined Use

Haemonchus contortus is recognized as one of the important health problems in small ruminants, leading to reduced production and economic loss for farmers worldwide. Prepatent diagnosis of H. contortus infection is crucial to improve control strategies as this helminth may remove up to one-fifth of total erythrocytes and may cause anemia, edema, diarrhea, and ultimately death in young animals. In this study, one of the excretory and secretory products, rHc-HCA59, was purified and used as antigen to detect specific antibodies in H. contortus infected goats during prepatent stage of infection using indirect enzyme linked immunosorbent assay (ELISA) as screening test. All goats (n = 38) were housed indoor, experimentally infected with 8000 infective larvae (L3) of H. contortus, and serum samples were collected prior to infection and at 14th day of infection. Immunoblotting was performed to confirm the results of indirect ELISA, evaluate the cross reactivity against rHc-HCA59 in sera of most common co-infecting parasites and rectify the false negative samples. Furthermore, three different batches of rHc-HCA59 were produced to evaluate the repeatability of ELISA. No eggs were detected in feces of all goats collected at 7th and 14th day of infection but, H. contortus eggs were detected at 21 days post infection in the feces. Indirect ELISA performed in this study showed 87% sensitivity and 100% specificity. The western blot analysis confirmed immunoreactivity in serum samples which scored positive in indirect ELISA and recognized the samples as negative which had OD450 lower than negative cut-off value in indirect ELISA. Furthermore, all false negative sera (n = 5) that had OD450 value between positive and negative cut-off value in rHc-HCA59 based ELISA were clearly positive in western blot. Moreover, no cross-reactivity was detected in ELISA and western blotting against rHc-HCA59 in positive sera of Toxoplasma gondii, Fasciola hepatica, and Trichinella spiralis. The results of this study concluded that combined use of indirect ELISA and western blotting with rHc-HCA59 is a potential immunodiagnostic tool for the detection of H. contortus infection during prepatent period in goats.

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Optimization and Validation of Indirect ELISA Using Truncated TssB Protein for the Serodiagnosis of Glanders amongst Equines

The Scientific World JOURNAL ◽

10.1155/2014/469407 ◽

2014 ◽

Vol 2014 ◽

pp. 1-6 ◽

Cited By ~ 8

Author(s):

Harisankar Singha ◽

Praveen Malik ◽

Sachin K. Goyal ◽

Sandip K. Khurana ◽

Chiranjay Mukhopadhyay ◽

...

Keyword(s):

Indirect Elisa ◽

Expression System ◽

Cross Reactivity ◽

Amino Acid Sequences ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Diagnostic Efficacy ◽

Diagnostic Potential ◽

Prokaryotic Expression System ◽

Control Serum

Objective. To express truncated TssB protein ofBurkholderia malleiand to evaluate its diagnostic efficacy for serological detection of glanders among equines.Materials and Methods. In an attempt to develop recombinant protein based enzyme-linked immunosorbent assay (ELISA), N-terminal 200 amino acid sequences ofB. malleiTssB protein—a type 6 secretory effector protein—were expressed in prokaryotic expression system. Diagnostic potential of recombinant TssB protein was evaluated in indirect ELISA using a panel of glanders positive (n=49), negative (n=30), and field serum samples (n=1811). Cross-reactivity of the assay was assessed with equine disease control serum and human melioidosis positive serum.Results. In comparison to CFT, diagnostic sensitivity and specificity of ELISA were 99.7% and 100%, respectively.Conclusions. The indirect ELISA method using the truncated TssB offered safer and more rapid and efficient means of serodiagnosis of glanders in equines. These data highlight the use of TssB as potential diagnostic antigen for serological diagnosis of glanders.

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Evaluation of Recombinant Antigens for Serodiagnosis of Chagas’ Disease in South and Central America

Journal of Clinical Microbiology ◽

10.1128/jcm.37.5.1554-1560.1999 ◽

1999 ◽

Vol 37 (5) ◽

pp. 1554-1560 ◽

Cited By ~ 80

Author(s):

Eufrosina S. Umezawa ◽

Sueli F. Bastos ◽

Mario E. Camargo ◽

Luci M. Yamauchi ◽

Márcia R. Santos ◽

...

Keyword(s):

Chagas Disease ◽

Recombinant Proteins ◽

High Sensitivity ◽

Cross Reactivity ◽

Recombinant Antigen ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Serological Tests ◽

Recombinant Antigens ◽

Fusion Products

The commercially available diagnostic tests for Chagas’ disease employ whole extracts or semipurified fractions ofTrypanosoma cruzi epimastigotes. Considerable variation in the reproducibility and reliability of these tests has been reported by different research laboratories, mainly due to cross-reactivity with other pathogens and standardization of the reagents. The use of recombinant antigens for the serodiagnosis of Chagas’ disease is recommended to increase the sensitivity and specificity of serological tests. Expressed in Escherichia coli, as fusion products with glutathione S-transferase, six T. cruzirecombinant antigens (H49, JL7, A13, B13, JL8, and 1F8) were evaluated in an enzyme-linked immunosorbent assay for Chagas’ disease. The study was carried out with a panel of 541 serum samples of chagasic and nonchagasic patients from nine countries of Latin America (Argentina, Bolivia, Brazil, Chile, Colombia, El Salvador, Guatemala, Honduras, and Venezuela). The optimal concentration of each recombinant antigen for coating of plates was determined with the help of125I-labelled recombinant proteins. While the specificity of the epimastigote antigen was 84% because of false positives from leishmaniasis cases, for the recombinant antigens it varied from 96.2 to 99.6%. Recombinant antigens reacted with 79 to 100% of serum samples from chronic chagasic patients. In this way, it is proposed that a mixture of a few T. cruzi recombinant antigens should be employed in a diagnostic kit to minimize individual variation and promote high sensitivity in the diagnosis of Chagas’ disease.

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Strongyloides-Specific IgE Phage cDNA Clones and Development of a Novel ELISA for Strongyloidiasis

Diagnostics ◽

10.3390/diagnostics11060985 ◽

2021 ◽

Vol 11 (6) ◽

pp. 985

Author(s):

Hussain Ahmad ◽

Norsyahida Arifin ◽

Thomas J. Nolan ◽

James B. Lok ◽

Nor Suhada Anuar ◽

...

Keyword(s):

Recombinant Protein ◽

Western Blot ◽

Rapid Test ◽

Strongyloides Stercoralis ◽

Specific Ige ◽

Diagnostic Value ◽

Epidemiological Studies ◽

Enzyme Linked Immunosorbent Assay ◽

Cdna Clones ◽

Diagnostic Potential

Strongyloidiasis, caused mainly by the nematode Strongyloides stercoralis, is prevalent worldwide and potentially fatal in immunosuppressed patients. We report on a new IgE biomarker to diagnose Strongyloides infection. Sera from two groups infected with Strongyloides served as positive samples: Group 1A, in which infection was confirmed by stool-microscopy and/or stool-polymerase chain reaction (PCR) and was seropositive by an IgG-enzyme linked immunosorbent assay (ELISA) and an IgG4 rapid test, and Group 1B in which infection was confirmed by stool-PCR but was seronegative. Negative samples (controls) comprised infections with other parasites (Group II) and healthy donors (Group III). Immunoscreenings of an S. stercoralis complementary DNA (cDNA) library were performed, and the cDNA clone with the highest diagnostic potential (clone A133) was selected for recombinant protein production and then evaluated using IgE Western blot and ELISA. The Western blot showed that the recombinant protein (rA133) was 100% reactive with Group IA (n = 10) and Group IB (n = 5), and 96% non-reactive with Groups II and III (n = 25). Subsequently, the IgE-ELISA was developed and showed 100% diagnostic sensitivity in Groups IA (n = 32) and IB (n = 11); and 99.3% specificity in Groups II and III (n = 144). In conclusion, this study has identified rA133 as a novel recombinant protein with potential diagnostic value, and that the IgE-ELISA incorporating this protein may be useful for patient diagnosis and epidemiological studies.

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Evaluation of a Western Blot Method for the Detection of Yersinia Antibodies: Evidence of Serological Cross-Reactivity between Yersinia Outer Membrane Proteins and Borrelia burgdorferi

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.12.11.1269-1274.2005 ◽

2005 ◽

Vol 12 (11) ◽

pp. 1269-1274 ◽

Cited By ~ 10

Author(s):

Mindy L. Rawlins ◽

Cecilia Gerstner ◽

Harry R. Hill ◽

Christine M. Litwin

Keyword(s):

Membrane Proteins ◽

Borrelia Burgdorferi ◽

Western Blot ◽

Outer Membrane ◽

Sensitivity And Specificity ◽

Reactive Arthritis ◽

Outer Membrane Proteins ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Western Blot Assay

ABSTRACT Yersinia enterocolitica and Yersinia pseudotuberculosis have been identified as causative organisms of reactive arthritis in humans. We evaluated a Western blot assay which uses Yersinia outer membrane proteins as antigens for the detection of Yersinia antibodies as a replacement for the complement fixation (CF) assay. Clinical agreement, sensitivity, and specificity were determined by testing 19 positive and 21 negative serum samples by the CF assay, Western blot assay, and enzyme-linked immunosorbent assay (ELISA). The CF assay and ELISA were compared to the Western blot assay, which was the reference method used in this study. Sera with antibodies that could potentially cross-react with Yersinia were also tested by the Western blot assay. The agreement, sensitivity, and specificity of the CF method were 61%, 26%, and 95%, respectively; and those for the ELISA were 89%, 95%, and 82%, respectively. The prevalences of Yersinia antibodies in 50 healthy donors were 6% for immunoglobulin G (IgG), 2% for IgA, and 2% for IgM. Sera positive for Bartonella henselae, Brucella, Chlamydia pneumoniae, and Rickettsia rickettsii antibodies showed cross-reactivity by the Western blot assay. The highest cross-reactivity was observed with Borrelia burgdorferi; 5 of 11 (45%) specimens were cross-reactive by the IgM-specific assay. Overall, the Western blot assay performs acceptably and is more sensitive than the CF assay, warranting replacement of the CF assay in the laboratory. Due to the evidence of cross-reactivity, particularly with B. burgdorferi, which can cause an oligoarthritis similar to reactive arthritis, the diagnosis of reactive arthritis should be based on clinical findings and complete serologic analysis of the potential causative infectious pathogens.

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Absence of Immunogenicity of Diaspirin Cross-Linked Hemoglobin in Humans

Blood ◽

10.1182/blood.v91.2.710 ◽

1998 ◽

Vol 91 (2) ◽

pp. 710-716 ◽

Cited By ~ 11

Author(s):

Mehul J. Patel ◽

Edwin J. Webb ◽

Tina E. Shelbourn ◽

Cynthia Mattia-Goldberg ◽

Andrew J.T. George ◽

...

Keyword(s):

Clinical Trials ◽

Western Blot ◽

Optical Density ◽

Oxygen Carrier ◽

Density Ratio ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Western Blot Assay ◽

Patient Sample ◽

Optical Density Ratio

Abstract Diaspirin cross-linked hemoglobin (DCLHb) is an intramolecularly cross-linked hemoglobin-based oxygen carrier being developed as a therapy for acute blood loss. We report here the absence of immunogenicity of DCLHb in patients enrolled in phase II and III clinical trials of DCLHb. Two very sensitive immunoassays, an enzyme-linked immunosorbent assay (ELISA) and a Western blot assay, were developed and validated for this assessment. The DCLHb-antibodies used in these assays were raised in monkeys, had similar affinities for DCLHb and native human hemoglobin (SFHb), and showed cross-reactivity for subunits of DCLHb and SFHb on the Western blot, suggesting that these antibodies were elicited as a xenogenic response to the protein. In the ELISA, the optical density of a patient sample exposed to DCLHb-coated wells was compared with that of the patient sample exposed to carbonate buffer-coated wells; an optical density ratio of 1.4 was established for discriminating between a positive (reactive) or negative DCLHb antibody response. To date, all of the more than 300 patient specimens (preinfusion and postinfusion) from clinical trials have exhibited a ratio of less than 1.4, confirming the lack of preexisting antibodies to DCLHb and clearly showing the absence of DCLHb antibodies after exposure to this new biologic entity. There has been no requirement for use of the confirmatory Western blot assay. Taken together, the results from this study indicate DCLHb is not immunogenic in humans at doses evaluated clinically.

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Demonstration of cross-reactive antigens in F38 and related mycoplasmas by enzyme-linked immunosorbent assay (ELISA) and immunoblotting

Journal of Hygiene ◽

10.1017/s002217240006232x ◽

1985 ◽

Vol 95 (1) ◽

pp. 95-106 ◽

Cited By ~ 13

Author(s):

M. Kanyi Kibe ◽

D. E. Bidwell ◽

P. Turp ◽

G. R. Smith

Keyword(s):

Immunosorbent Assay ◽

Immunoblot Analysis ◽

Cross Reactivity ◽

Mycoplasma Species ◽

Enzyme Linked Immunosorbent Assay ◽

Major Protein ◽

Protein Antigens ◽

Contagious Caprine Pleuropneumonia ◽

Mycoplasma Mycoides ◽

Immunoblotting Technique

SUMMARYThe ELISA and an immunoblotting technique were used to study F38-type mycoplasmas – an important cause of contagious caprine pleuropneumonia – and a number of related mycoplasma species, subspecies, types or serogroups.Two-way ELISA cross-reactivity was demonstrated between five mycoplasmas, namely strain F38,Mycoplasma mycoidessubsp.mycoides(LC strain),M. equigenilalium, M. primatumand bovine serogroup 7. In addition one-way cross-reactivity was demonstrated between F38 and each of the following mycoplasmas:M. mycoidessubsp.mycoides(two SC strains),M. mycoidessubsp.capri, and bovine serogroup L. F38 andM. capricolumdid not cross-react.Immunoblot analysis, unlike ELISA, revealed that F38 andM. capricolumwere closely related. At least four major protein antigens were shared between F38,M. mycoidessubsp.mycoides(SC and LC strains),M. mycoidessubsp.capriand bovine serogroup 7. The ELISA cross-reactions (above) shown byM. equigenitaliumandM. primatumwith each other, with F38 and with other mycoplasmas were not apparent by immunoblotting.

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Decorin Binding Proteins A and B in the Serodiagnosis of Lyme Disease in North America

Clinical and Vaccine Immunology ◽

10.1128/cvi.00383-14 ◽

2014 ◽

Vol 21 (10) ◽

pp. 1426-1436 ◽

Cited By ~ 9

Author(s):

Paul M. Arnaboldi ◽

Mariya Sambir ◽

Raymond J. Dattwyler

Keyword(s):

Lyme Disease ◽

Protein A ◽

Laboratory Diagnosis ◽

Cross Reactivity ◽

Antibody Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Protein Antigens ◽

Content Type ◽

Linear Epitopes ◽

The Individual

ABSTRACTThe laboratory diagnosis of Lyme disease is based upon the detection of antibodies generated againstBorrelia burgdorferiusing a two-tier assay, typically consisting of an enzyme-linked immunosorbent assay (ELISA), followed by a Western blot. This system, put into place to address the nonspecificity associated with standalone first-tier assays, is insensitive for diagnosing early infection, when most people seek care. The use of bacterial lysates or whole-protein antigens as first-tier assay targets contributes to nonspecificity due, in part, to the presence of cross-reactive epitopes that are also found in other bacteria. This precludes their use as sensitive standalone assays. The use of peptides containing linear epitopes that are highly specific forB. burgdorferioffers a method for reducing this cross-reactivity. In the present study, we mapped the linear epitopes of the prominently expressedBorreliaadhesins decorin binding protein A (DbpA) and DbpB. We identified several epitopes in each protein that were highly conserved among North American strains ofB. burgdorferi, and we screened peptides containing specific epitopes using serum panels from early and late Lyme disease patients. The individual peptides primarily detected IgM but not IgG, while the proteins efficiently detected both IgM and IgG. While no individual peptide demonstrated better utility for antibody detection than its respective whole protein, an assay containing a combination of a DbpA and a DbpB peptide adequately detected both IgM and IgG, accurately identifying 87.5% (84/96) of the early Lyme disease patients and 80.0% (16/20) of the late Lyme disease patients.

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Evaluation of salivary biomarkers for the diagnosis of periodontitis

BMC Oral Health ◽

10.1186/s12903-021-01600-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yong Zhang ◽

Ni Kang ◽

Fei Xue ◽

Jing Qiao ◽

Jinyu Duan ◽

...

Keyword(s):

Periodontal Disease ◽

Healthy Subjects ◽

Type I Collagen ◽

Area Under The Curve ◽

Diagnostic Value ◽

Enzyme Linked Immunosorbent Assay ◽

Type I ◽

Cross Sectional ◽

Diagnostic Potential ◽

Auc Value

Abstract Background Salivary interleukin (IL)-1β, matrix metalloproteinase (MMP)-8, pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP) and Porphyromonas gingivalis (Pg) are related to periodontitis. This study aimed to investigate the diagnostic potential of these biomarkers and to build a prediction panel for diagnosing periodontal disease. Methods A total of 80 participants were enrolled in a cross-sectional study and divided into healthy (n = 25), gingivitis (n = 24), and periodontitis (n = 31) groups based on their periodontal exam results. A full mouth periodontal examination was performed and unstimulated saliva was collected. Salivary IL-1β, MMP-8, ICTP, and Pg were assessed using enzyme-linked immunosorbent assay (ELISA) and quantitative real time PCR (qPCR). Their potentials for diagnosing periodontal disease were analyzed and combined prediction panels of periodontal disease were evaluated. Results As a single marker, IL-1β showed the best diagnostic value of the four markers evaluated and exhibited an area under the curve (AUC) value of 0.88 with 90% sensitivity and 76% specificity for discriminating periodontitis subjects from healthy subjects, an AUC value of 0.80 with 83% sensitivity and 76% specificity for discriminating gingivitis subjects from healthy subjects and an AUC value of 0.66 with 68% sensitivity and 64% specificity for differentiating periodontitis subjects from gingivitis subjects. The combination of IL-1β, ICTP, and Pg exhibited the highest efficacy for discriminating periodontitis subjects from healthy subjects (AUC = 0.94) and gingivitis subjects (AUC = 0.77). The combination of IL-1β and MMP-8 exhibited the best ability to discriminate gingivitis from healthy subjects (AUC = 0.84). Conclusions Salivary IL-1β, MMP-8, ICTP, and Pg showed significant effectiveness for diagnosing periodontal disease. The combination of IL-1β, ICTP, and Pg can be used to discriminate periodontitis subjects from healthy subjects and gingivitis subjects, and the combination of IL-1β and MMP-8 can be used to discriminate gingivitis subjects from healthy subjects.

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