Evaluation of a NovelAspergillusAntigen Enzyme-Linked Immunosorbent Assay

ABSTRACTInvasive aspergillosis (IA) is a life-threatening infection that mainly occurs in immunocompromised patients. Here, we compared the novelAspergillus-specific galactomannoprotein (GP) enzyme-linked immunosorbent assay (ELISA) (Euroimmun Medizinische Labordiagnostika AG) to the established PlateliaAspergillusgalactomannan (GM) ELISA (Bio-Rad Laboratories) for the detection of IA. A total of 267 serum samples from 45 cases of proven and 4 episodes of probable IA (according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group [EORTC/MSG] criteria) and 156 sera from patients without evidence of IA were tested. Pearson’s correlation statistics, as well as sensitivity and specificity, were calculated using manufacturer-recommended (GM) or optimized (GP) cutoff levels.Aspergillus fumigatuswas found in 88% of culture-positive infections. When we analyzed all 423 serum samples, GM and GP tests correlated strongly (r = 0.82,P < 0.0001). Among proven IA cases using samples obtained as closely as possible to the day of proven diagnosis, the sensitivity for both tests was 40%. All cases of probable IA (defined by positive GM testing) were also GP positive. Concordant results of the two ELISAs were obtained in 43 of 49 samples (88%). Extending measurements to all sera available in the time frame of 7 days prior to 7 days after the day of proven diagnosis, 47% and 56% of the cases were detected by the GM and GP tests, respectively. Specificity was 99% for GM and 96% for GP testing. For the diagnosis of IA, sensitivity and specificity of the novel GP ELISA are similar to those of the Platelia GM ELISA. The low sensitivities of both tests underline the need for serial testing in patients at risk for IA.

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Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay

Clinical and Vaccine Immunology ◽

10.1128/cvi.00516-12 ◽

2012 ◽

Vol 20 (1) ◽

pp. 9-16 ◽

Cited By ~ 28

Author(s):

Neekun Sharma ◽

Akitoyo Hotta ◽

Yoshie Yamamoto ◽

Osamu Fujita ◽

Akihiko Uda ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Francisella Tularensis ◽

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

The Novel ◽

Serum Samples ◽

Content Type ◽

Microagglutination Test ◽

Highly Sensitive ◽

High Degree

ABSTRACTA novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies againstFrancisella tularensisin humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed againstF. tularensislipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivatedF. tularensis-immunized rabbits, andF. tularensis-infected mice. Antibodies againstF. tularensiswere successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2= 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species.

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Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia

Journal of Clinical Microbiology ◽

10.1128/jcm.03259-15 ◽

2016 ◽

Vol 54 (6) ◽

pp. 1557-1565 ◽

Cited By ~ 1

Author(s):

Martin Heller ◽

Nimmo Gicheru ◽

Georgina Tjipura-Zaire ◽

Cecilia Muriuki ◽

Mingyan Yu ◽

...

Keyword(s):

Immunosorbent Assay ◽

Rapid Test ◽

Lateral Flow ◽

Sub Saharan Africa ◽

Enzyme Linked Immunosorbent Assay ◽

Contagious Bovine Pleuropneumonia ◽

Serum Samples ◽

Content Type ◽

Mycoplasma Mycoides ◽

Sub Saharan

Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease that is widespread in sub-Saharan Africa. It is caused byMycoplasma mycoidessubsp.mycoides, a bacterium belonging to theMycoplasma mycoidescluster. In the absence of an efficient CBPP vaccine, improved and easy-to-use diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa. Here we describe the comprehensive screening of 17 immunogenicMycoplasma mycoidessubsp.mycoidesproteins using well-characterized bovine sera for the development of a novel cocktail enzyme-linked immunosorbent assay (ELISA) for laboratory use. Two recombinantMycoplasmaimmunogens, MSC_0136 and MSC_0636, were used to set up a standardized cocktail ELISA protocol. According to the results from more than 100 serum samples tested, the sensitivity and specificity of the novel cocktail ELISA were 85.6% and 96.4%, respectively, with an overall diagnostic accuracy comparable to that of the Office International des Epizooties (OIE)-prescribed serological assays. In addition, we provide a proof of principle for a field-applicable, easy-to-use commercially produced prototype lateral-flow test for rapid (<30-min) diagnosis of CBPP.

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Diagnosis of Visceral Leishmaniasis by Enzyme-Linked Immunosorbent Assay Using Urine Samples

Clinical and Vaccine Immunology ◽

10.1128/cdli.9.4.789-794.2002 ◽

2002 ◽

Vol 9 (4) ◽

pp. 789-794 ◽

Cited By ~ 3

Author(s):

Mohammad Zahidul Islam ◽

Makoto Itoh ◽

S. M. Shamsuzzaman ◽

Rusella Mirza ◽

Farzana Matin ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Sensitivity And Specificity ◽

Immunosorbent Assay ◽

Leishmania Donovani ◽

Laboratory Diagnosis ◽

Urine Samples ◽

Enzyme Linked Immunosorbent Assay ◽

Healthy Controls ◽

Serum Samples ◽

Crude Antigen

ABSTRACT A diagnostic method has been developed to detect anti-Leishmania donovani immunoglobulin G (IgG) in urine by enzyme-linked immunosorbent assay (ELISA). In measuring anti-L. donovani IgG, IgA, and IgM in urine, the method performed best in the detection of IgG. The sensitivity and specificity of the assay were determined with panels of urine samples from 62 visceral leishmaniasis (VL) patients, 59 healthy controls from areas of endemicity, 53 healthy controls from areas of nonendemicity, 59 malaria patients, 13 tuberculosis patients, 23 cutaneous leishmaniasis patients, and 7 patients with other diseases. Using L. donovani promastigote crude antigen, the test had 93.5% sensitivity (58 positives of 62 VL patient samples) and 89.3% specificity (191 negatives of 214 non-VL patient samples). The ELISA with acetone-treated L. donovani promastigote antigen raised the sensitivity and specificity to 95.0 and 95.3%, respectively. Western blot analysis revealed that most of the samples that cross-reacted with crude antigen in ELISA did not recognize any antigenic component of L. donovani crude antigen. We also checked 40 serum samples from the same group of VL patients for anti-L. donovani IgG and got 90.0% sensitivity with both crude and acetone-treated antigens. As collection of urine is much easier than collection of serum, the detection of anti-L. donovani IgG in urine with acetone-treated antigen will be useful in epidemiological studies. It could be an adjunct of laboratory diagnosis.

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Evaluation of a Blocking ELISA Using a Urease Conjugate for the Detection of Antibodies to Pseudorabies Virus

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870001200313 ◽

2000 ◽

Vol 12 (3) ◽

pp. 266-268 ◽

Cited By ~ 3

Author(s):

Maria Gabriela Echeverría ◽

Edgardo Omar Nosetto ◽

Maria Elisa Etcheverrigaray

Keyword(s):

Sensitivity And Specificity ◽

Immunosorbent Assay ◽

Pseudorabies Virus ◽

Visual Assessment ◽

Field Conditions ◽

Virus Neutralization ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Blocking Elisa ◽

Elisa Format

A blocking enzyme-linked immunosorbent assay (ELISA) using a urease conjugate (U-B-ELISA) was evaluated for screening sera for antibodies to pseudorabies virus under field conditions. A total of 764 serum samples were analyzed by U-B-ELISA. Of these, 264 were evaluated by both virus neutralization and U-B-ELISA, and the results were compared. U-B-ELISA showed 98.5% and 98.9% sensitivity and specificity, respectively. This test combines the sensitivity and specificity of the blocking ELISA format while allowing visual assessment of results.

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Development of an Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Ringworm Infection in Cattle

Clinical and Vaccine Immunology ◽

10.1128/cvi.00243-13 ◽

2013 ◽

Vol 20 (8) ◽

pp. 1150-1154 ◽

Cited By ~ 6

Author(s):

Elena Tatiana Băguţ ◽

Ludivine Cambier ◽

Marie-Pierre Heinen ◽

Vasile Cozma ◽

Michel Monod ◽

...

Keyword(s):

Positive Predictive Value ◽

Negative Predictive Value ◽

Immunosorbent Assay ◽

Predictive Value ◽

Skin Lesions ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Predictive Values ◽

Content Type ◽

Ringworm Infection

ABSTRACTThe aim of this study was to develop an in-house enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of ringworm infection in cattle. We used available recombinant forms ofTrichophyton rubrumdipeptidyl peptidase V (TruDppV) andT. rubrumleucin aminopeptidase 2 (TruLap2), which are 98% identical toTrichophyton verrucosumorthologues. Field serum samples from 135 cattle with ringworm infection, as confirmed by direct microscopy, fluorescence microscopy, and PCR, and from 55 cattle without any apparent skin lesions or history of ringworm infection that served as negative controls were used. Sensitivities, specificities, and positive and negative predictive values were determined to evaluate the diagnostic value of our ELISA. Overall, the ELISAs based on recombinant TruDppV and TruLap2 discriminated well between infected animals and healthy controls. Highly significant differences (P< 0.0001, Mann-Whitney U test) were noted between optical density values obtained when sera from infected versus control cattle were tested. The ELISA developed for the detection of specific antibodies against DppV gave 89.6% sensitivity, 92.7% specificity, a 96.8% positive predictive value, and a 78.4% negative predictive value. The recombinant TruLap2-based ELISA displayed 88.1% sensitivity, 90.9% specificity, a 95.9% positive predictive value, and a 75.7% negative predictive value. To the best of our knowledge, this is the first ELISA based on recombinant antigens for assessing immune responses to ringworm infection in cattle; it is particularly suitable for epidemiological studies and also for the evaluation of vaccines and/or vaccination procedures.

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Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting Serum Antibodies against Mycobacterium avium subsp. paratuberculosis in Fallow Deer

Clinical and Vaccine Immunology ◽

10.1128/cvi.00159-14 ◽

2014 ◽

Vol 21 (8) ◽

pp. 1077-1085 ◽

Cited By ~ 8

Author(s):

José M. Prieto ◽

Ana Balseiro ◽

Rosa Casais ◽

Naiara Abendaño ◽

Liam E. Fitzgerald ◽

...

Keyword(s):

Immunosorbent Assay ◽

Mycobacterium Avium ◽

Cost Effective ◽

Fallow Deer ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Igg Antibody ◽

Tissue Samples ◽

Content Type ◽

Mycobacterium Avium Subsp Paratuberculosis

ABSTRACTThe enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies againstMycobacterium aviumsubsp.paratuberculosisin wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deerM. aviumsubsp.paratuberculosisisolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detectM. aviumsubsp.paratuberculosisantibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations.

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Evaluation of an in-house indirect enzyme-linked immunosorbent assay of feline panleukopenia VP2 subunit antigen in comparison to hemagglutination inhibition assay to monitor tiger antibody levels by Bayesian approach

10.21203/rs.3.rs-23779/v1 ◽

2020 ◽

Author(s):

Chanakan Areewong ◽

Amarin Rittipornlertrak ◽

Boondarika Nambooppha ◽

Itsarapan Fhaikrue ◽

Tawatchai Singhla ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Immunosorbent Assay ◽

Hemagglutination Inhibition ◽

Indirect Elisa ◽

Enzyme Linked Immunosorbent Assay ◽

Panthera Tigris ◽

Serum Samples ◽

Feline Panleukopenia Virus ◽

Elisa Testing ◽

Antibody Levels

Abstract Background: Feline panleukopenia virus (FPV) is an etiologic pathogen of feline panleukopenia that infects all members of Felidae including tigers (Panthera tigris). Vaccination against FPV among wild felid species has long been practiced in zoos worldwide. However, few studies have assessed tiger immune response post-vaccination due to the absence of a serological diagnostic tool. To address these limitations, this study aimed to develop an in-house indirect enzyme-linked immunosorbent assay (ELISA) for the monitoring of tiger antibody levels against the feline panleukopenia vaccine by employing the synthesized subunit capsid protein VP2. An in-house horseradish peroxidase (HRP) conjugated rabbit anti-tiger immunoglobulin G (IgG) polyclonal antibody (HRP-anti-tiger IgG) was produced in this study and employed in the assay. It was then compared to a commercial HRP-conjugated goat anti-cat IgG (HRP-anti-cat IgG). Sensitivity and specificity were evaluated using the Bayesian model with conditional dependence being assumed between both HRP-conjugated antibody-based ELISAs and hemagglutination-inhibition (HI) tests. Results: The posterior estimates for sensitivity and specificity of two indirect ELISA HRP-conjugated antibodies were higher than those of the HI test. The sensitivity and specificity of indirect ELISA for HRP-anti-tiger IgG and HRP-anti-cat IgG were 86.5%, 57.2% and 86.7%, 64.6%, respectively, while the results of the HI test were 79.1 and 54.1%. In applications, 89.6% (198/221) of tiger serum samples were determined to be seropositive by indirect ELISA testing. Conclusion: The results support evidence that an in-house indirect ELISA developed in this study could be used as a serological tool for the effective detection of tiger antibodies.

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Enzyme-Linked Immunosorbent Assay Using Recombinant SAG1 Antigen To Detect Toxoplasma gondii-Specific Immunoglobulin G Antibodies in Human Sera and Saliva

Clinical and Vaccine Immunology ◽

10.1128/cvi.00512-12 ◽

2013 ◽

Vol 20 (4) ◽

pp. 468-473 ◽

Cited By ~ 16

Author(s):

Nouha Chahed Bel-Ochi ◽

Aïda Bouratbine ◽

Mohamed Mousli

Keyword(s):

Toxoplasma Gondii ◽

Sensitivity And Specificity ◽

Specific Antigen ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Native Form ◽

Content Type ◽

Percent Agreement ◽

Human Sera ◽

Commercial Kits

ABSTRACTSerologic detection ofToxoplasma gondiiIgG antibodies is widely accepted as a means to determine immune status and susceptibility toToxoplasmainfection during pregnancy. However, current commercial kits present some drawbacks, such as a requirement for whole-parasite antigen preparation or interassay variability. To address these problems, the purpose of this study was to produce a whole sequence of the recombinantT. gondiiSAG1 antigen (rSAG1) to assess its diagnostic performance inToxoplasmaIgG screening and to explore a saliva-based method as a noninvasive alternative to serum-based testing. rSAG1 was expressed in recombinant bacteria as inclusion bodies, purified through one-step affinity chromatography, and refolded in native form by dialysis. A large amount was obtained, and the specific antigen immunoreactivity was confirmed by immunoblotting. Two rSAG1-based enzyme-linked immunosorbent assays (ELISAs) applied to paired serum and saliva samples were designed. The rSAG1-based ELISA evaluation consisted of testing intrinsic sensitivity and specificity of 49 serum samples from patients immune to toxoplasmosis and 42 serum samples from nonimmune controls identified by routinely used kits. To assess agreement between serum-based and saliva-based tests, the positive percent agreement (PPA) and negative percent agreement (NPA) between the 2 tests were estimated. The rSAG1 serum-based ELISA detected specific IgG with 100% sensitivity and specificity. The PPA and NPA between the serum-based and saliva-based tests varied according to the selected optical density threshold in saliva. Thus, for a selected cutoff of 0.14, the PPA was 100% and the NPA was 88.1%, whereas for a selected cutoff of 0.29, the PPA was 67.3% and the NPA was 100%.

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Recombinant Antigen-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Baylisascaris procyonis Larva Migrans

Clinical and Vaccine Immunology ◽

10.1128/cvi.00083-11 ◽

2011 ◽

Vol 18 (10) ◽

pp. 1650-1655 ◽

Cited By ~ 14

Author(s):

Sriveny Dangoudoubiyam ◽

Ramesh Vemulapalli ◽

Momar Ndao ◽

Kevin R. Kazacos

Keyword(s):

Immunosorbent Assay ◽

Cross Reactivity ◽

Larva Migrans ◽

Enzyme Linked Immunosorbent Assay ◽

Parasitic Infections ◽

Serum Samples ◽

Western Blot Assay ◽

Human Patient ◽

Content Type ◽

Baylisascaris Procyonis

ABSTRACTBaylisascarislarva migrans is an important zoonotic disease caused byBaylisascaris procyonis, the raccoon roundworm, and is being increasingly considered in the differential diagnosis of eosinophilic meningoencephalitis in children and young adults. Although aB. procyonisexcretory-secretory (BPES) antigen-based enzyme-linked immunosorbent assay (ELISA) and a Western blot assay are useful in the immunodiagnosis of this infection, cross-reactivity remains a major problem. Recently, a recombinantB. procyonisantigen, BpRAG1, was reported for use in the development of improved serological assays for the diagnosis ofBaylisascarislarva migrans. In this study, we tested a total of 384 human patient serum samples in a BpRAG1 ELISA, including samples from 20 patients with clinicalBaylisascarislarva migrans, 137 patients with other parasitic infections (8 helminth and 4 protozoan), and 227 individuals with unknown/suspected parasitic infections. A sensitivity of 85% and a specificity of 86.9% were observed with the BpRAG1 ELISA, compared to only 39.4% specificity with the BPES ELISA. In addition, the BpRAG1 ELISA had a low degree of cross-reactivity with antibodies toToxocarainfection (25%), while the BPES antigen showed 90.6% cross-reactivity. Based on these results, the BpRAG1 antigen has a high degree of sensitivity and specificity and should be very useful and reliable in the diagnosis and seroepidemiology ofBaylisascarislarva migrans by ELISA.

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Comparison of Performance Characteristics of Aspergillus PCR in Testing a Range of Blood-Based Samples in Accordance with International Methodological Recommendations

Journal of Clinical Microbiology ◽

10.1128/jcm.02814-15 ◽

2016 ◽

Vol 54 (3) ◽

pp. 705-711 ◽

Cited By ~ 16

Author(s):

Jan Springer ◽

P. Lewis White ◽

Shanna Hamilton ◽

Denise Michel ◽

Rosemary A. Barnes ◽

...

Keyword(s):

Whole Blood ◽

Clinical Performance ◽

Fungal Infections ◽

Plasma Samples ◽

Serum Samples ◽

European Organization ◽

Content Type ◽

Extraction Processes ◽

Automated Dna Extraction ◽

Control Study

Standardized methodologies for the molecular detection of invasive aspergillosis (IA) have been established by the EuropeanAspergillusPCR Initiative for the testing of whole blood, serum, and plasma. While some comparison of the performance ofAspergillusPCR when testing these different sample types has been performed, no single study has evaluated all three using the recommended protocols. StandardizedAspergillusPCR was performed on 423 whole-blood pellets (WBP), 583 plasma samples, and 419 serum samples obtained from hematology patients according to the recommendations. This analysis formed a bicenter retrospective anonymous case-control study, with diagnosis according to the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (11 probable cases and 36 controls). Values for clinical performance using individual and combined samples were calculated. For all samples, PCR positivity was significantly associated with cases of IA (for plasma,P= 0.0019; for serum,P= 0.0049; and for WBP,P= 0.0089). Plasma PCR generated the highest sensitivity (91%); the sensitivities for serum and WBP PCR were 80% and 55%, respectively. The highest specificity was achieved when testing WBP (96%), which was significantly superior to the specificities achieved when testing serum (69%,P= 0.0238) and plasma (53%,P= 0.0002). No cases were PCR negative in all specimen types, and no controls were PCR positive in all specimens. This study confirms thatAspergillusPCR testing of plasma provides robust performance while utilizing commercial automated DNA extraction processes. Combining PCR testing of different blood fractions allows IA to be both confidently diagnosed and excluded. A requirement for multiple PCR-positive plasma samples provides similar diagnostic utility and is technically less demanding. Time to diagnosis may be enhanced by testing multiple contemporaneously obtained sample types.

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