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

2013 ◽  
Vol 20 (4) ◽  
pp. 468-473 ◽  
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%.

scholarly journals Taenia saginata Metacestode Antigenic Fractions without Affinity to Concanavalin A Are an Important Source of Specific Antigens for the Diagnosis of Human Neurocysticercosis

2010 ◽  
Vol 17 (4) ◽  
pp. 638-644 ◽  
Author(s):  
Heliana B. Oliveira ◽  
Gleyce A. Machado ◽  
José R. Mineo ◽  
Julia M. Costa-Cruz
Keyword(s):  
Sensitivity And Specificity ◽  
Concanavalin A ◽  
Taenia Solium ◽  
Specific Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Taenia Saginata ◽  
Serum Samples ◽  
Unbound Fraction ◽  
Immunoblot Assay ◽  
Homologous Antigen

ABSTRACT Taenia saginata metacestode antigens have been constituted a useful alternative antigen for neurocysticercosis (NC) serodiagnosis, particularly due to an increasing difficulty to obtain Taenia solium homologous antigen. Cross-reactivity with Echinococcus granulosus infection occurs in homologous and heterologous antigens and could be avoided by using different purified methods. The present study evaluated antigen fractions obtained from saline extracts of T. saginata metacestodes purified by affinity chromatography with jacalin or concanavalin A (ConA) lectins to detect IgG antibodies by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis to diagnose human NC. Serum samples were collected from 142 individuals: 40 of them were diagnosed with NC, 62 presented Taenia sp. and other parasites, and 40 were apparently healthy individuals. The jacalin- and ConA-unbound fractions demonstrated sensitivity and specificity higher than those of bound fractions. Among unbound fractions, ConA demonstrated statistically higher sensitivity and specificity by ELISA (90% and 93.1%, respectively). By immunoblot assay, the 64- to 68-kDa component from the ConA-unbound fraction showed 100% sensitivity and specificity, making this component suitable for use as a specific antigen for diagnosis of NC. To our knowledge, this is the first report showing the relevance of using the unbound ConA fraction of T. saginata metacestodes to diagnose NC. In conclusion, the results obtained herein clearly demonstrate that antigenic fractions without affinity to ConA, obtained from T. saginata metacestodes, are an important source of specific peptides and are efficient in the diagnosis of NC when tested by immunoblot assay.

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

2012 ◽  
Vol 20 (1) ◽  
pp. 9-16 ◽  
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.

scholarly journals Effects of Pretreating Serum Samples on the Performance of a Latex Agglutination Test for Serodiagnosis of Paracoccidioidomycosis

2011 ◽  
Vol 19 (3) ◽  
pp. 386-390 ◽  
Author(s):  
Fabíola Silveira-Gomes ◽  
Silvia Helena Marques-da-Silva
Keyword(s):  
Sensitivity And Specificity ◽  
Bacterial Infections ◽  
Paracoccidioides Brasiliensis ◽  
Latex Agglutination ◽  
Agglutination Test ◽  
Latex Agglutination Test ◽  
Serum Samples ◽  
Content Type ◽  
Human Sera ◽  
Normal Human

ABSTRACTParacoccidioidomycosis (PCM) is a fungal disease caused byParacoccidioides brasiliensis, and Brazil is one of the principal countries where it is endemic. Diagnosis is based on the observation of buddingP. brasiliensisyeast in clinical specimens from patients; however, the sensitivity of the visualization of fungi is low, indicating that serological tests are used for early diagnosis. The double-immunodiffusion test (ID) is the “gold standard” test for serology in PCM, although the execution of this test requires the availability of laboratorial infrastructure. We report the improved performance of a latex agglutination test (LAT) by pretreating 30 serum samples from PCM patients and 71 controls (histoplasmosis and aspergillosis patients, patients with bacterial infections, and normal human sera) with a dilution buffer incubated at 37°C for 30 min. The sensitivity and specificity of the LAT test in the nonpretreated samples were 73% and 79%, respectively. However, when samples were pretreated, the sensitivity and specificity of the test increased to 90%. In this study, we did not observe cross-reactivity with histoplasmosis patient sera, but some reactions to sera from patients with aspergillosis and bacterial infections were noted. Normal human sera were not reactive in our tests. These results indicate the need for the elimination of heterologous reactions so that we can adequately use this method for screening cases of PCM.

scholarly journals Evaluation of Recombinant Dense Granule Antigen 7 (GRA7) of Toxoplasma gondii for Detection of Immunoglobulin G Antibodies and Analysis of a Major Antigenic Domain

1999 ◽  
Vol 6 (1) ◽  
pp. 24-29 ◽  
Author(s):  
Dirk Jacobs ◽  
Martine Vercammen ◽  
Eric Saman
Keyword(s):  
Monoclonal Antibody ◽  
Toxoplasma Gondii ◽  
Immunoglobulin G ◽  
Chronic Phase ◽  
Dense Granule ◽  
Leader Peptide ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Human Sera ◽  
Antigenic Domain

ABSTRACT Dense granule protein 7 (GRA7) of Toxoplasma gondii was expressed in Escherichia coli as a fusion protein. The leader peptide contained a 25-amino-acid mouse tumor necrosis factor fragment and six histidyl residues. After purification by metal chelate affinity chromatography, the antigen was evaluated in an enzyme-linked immunosorbent assay for detection of immunoglobulin G (IgG). For two sets of IgG-positive human serum samples, obtained from routine screening, an overall sensitivity of 81% was obtained. For chronic-phase sera, the sensitivity of detection was 79%, but chronic-phase sera with low titers were more difficult to detect (65% sensitivity for sera with immunofluorescence titer of 1/64). When GRA7 was combined with Tg34AR (rhoptry protein 2 C-terminal fragment), the sensitivity rose to 96%. For a set of acute-phase serum samples tested on GRA7, the sensitivity of detection was 94%, and high-titer IgM-positive sera were detected at an especially high rate. In contrast, when Tg34AR was used, the sensitivity was only 85% for this latter set of serum samples. Three truncated GRA7 fragments containing the same leader peptide as that of recombinant GRA7 were produced. The shortest fragment (97 N-terminal amino acids) was not reactive with human sera or with a specific anti-GRA7 monoclonal antibody, while the two larger fragments were reactive. The most important antigenic domain of GRA7 for human sera was localized between residues 97 and 146. The epitope for the specific monoclonal antibody could be further narrowed down by the use of synthetic peptides, but this epitope is not recognized by sera from T. gondii-infected humans. These results indicate that GRA7 may be considered as an additional tool for studying the immune response to T. gondii.

scholarly journals A New MIC1-MAG1 Recombinant Chimeric Antigen Can Be Used Instead of the Toxoplasma gondii Lysate Antigen in Serodiagnosis of Human Toxoplasmosis

2011 ◽  
Vol 19 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Lucyna Holec-Gąsior ◽  
Bartłomiej Ferra ◽  
Dorota Drapała ◽  
Dariusz Lautenbach ◽  
Józef Kur
Keyword(s):  
Toxoplasma Gondii ◽  
Expression System ◽  
Chimeric Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Content Type ◽  
Metal Affinity ◽  
Positive Serum ◽  
First Time ◽  
Microneme Protein

ABSTRACTThis study presents an evaluation of the MIC1 (microneme protein 1)-MAG1 (matrix antigen 1)Toxoplasma gondiirecombinant chimeric antigen for the serodiagnosis of human toxoplasmosis for the first time. The recombinant MIC1-MAG1 antigen was obtained as a fusion protein containing His tags at the N- and C-terminal ends using anEscherichia coliexpression system. After purification by metal affinity chromatography, the chimeric protein was tested for usefulness in an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-T. gondiiimmunoglobulin G (IgG). One hundred ten sera from patients at different stages of infection and 40 sera from seronegative patients were examined. The results obtained for the MIC1-MAG1 chimeric antigen were compared with those of IgG ELISAs using aToxoplasmalysate antigen (TLA), a combination of recombinant antigens (rMIC1ex2-rMAG1) and single recombinant proteins (rMIC1ex2 and rMAG1). The sensitivity of the IgG ELISA calculated from all of the positive serum samples was similar for the MIC1-MAG1 chimeric antigen (90.8%) and the TLA (91.8%), whereas the sensitivities of the other antigenic samples used were definitely lower, at 69.1% for the mixture of antigens, 75.5% for the rMIC1ex2, and 60% for rMAG1. This study demonstrates that the MIC1-MAG1 recombinant chimeric antigen can be used instead of the TLA in the serodiagnosis of human toxoplasmosis.

scholarly journals Development of an Immunochromatographic Assay Based on Dense Granule Protein 7 for Serological Detection of Toxoplasma gondii Infection

2013 ◽  
Vol 20 (4) ◽  
pp. 596-601 ◽  
Author(s):  
Mohamad Alaa Terkawi ◽  
Kyohko Kameyama ◽  
Nazim Hamza Rasul ◽  
Xuean Xuan ◽  
Yoshifumi Nishikawa
Keyword(s):  
Toxoplasma Gondii ◽  
Neospora Caninum ◽  
Latex Agglutination ◽  
Dense Granule ◽  
Diagnostic Tools ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Content Type ◽  
Specificity And Sensitivity ◽  
Field Samples

ABSTRACTDense granule antigen proteins derived fromToxoplasma gondii(TgGRAs) are potential antigens for the development of diagnostic tools. TgGRA7 and TgGRA14 were detected in the peritoneal fluid ofT. gondii-infected mice, suggesting that TgGRAs may be highly antigenic proteins. Here, TgGRA7 and TgGRA14 were evaluated as candidates for the development of a marker for a rapid diagnostic test. The specificity and sensitivity of purified recombinant proteins of TgGRA7 and TgGRA14 were compared in an indirect enzyme-linked immunosorbent assay (iELISA) using a series of serum samples fromT. gondii-experimentally infected mice and using recombinantT. gondiimajor surface antigen 2 (TgSAG2) as a reference control. The iELISA with TgGRA7 showed the greatest diagnostic accuracy and could detect anti-TgGRA7 antibody in acute and chronic infections. A total of 59 field samples from pigs were also examined by the iELISAs, and the results compared with those of the latex agglutination test (LAT). Among the three recombinant antigens, TgGRA7 had the highest rates of positivity, with significant concordance (88.14) and kappa value (0.76) in comparison with the results using LAT. Furthermore, an immunochromatographic test (ICT) based on recombinant TgGRA7 was developed for rapid detection of antibodies to the infection. The ICT differentiated clearly between sera fromT. gondii-infected mice and uninfected orNeospora caninum-infected mice. Pig sera were examined with the ICT, and the results compared favorably with those of LAT and iELISA for TgGRA7, with kappa values of 0.66 and 0.70 to 0.79, respectively. These data suggest that the ICT based on TgGRA7 is a promising diagnostic tool for routine testing in the clinic and mass screening of samples in the field.

scholarly journals Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats

2020 ◽  
Author(s):  
Charoonluk Jirapattharasate ◽  
Ruenruetai Udonsom ◽  
Apichai Prachasuphap ◽  
Kodcharad Jongpitisub ◽  
Panadda Dhepakson
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Protein ◽  
Sensitivity And Specificity ◽  
Western Blotting ◽  
Sodium Dodecyl ◽  
Laboratory Animals ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Field Samples ◽  
Specific Igg

Abstract Background The development of sensitive and specific methods for detecting Toxoplasma gondii infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently, various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in recombinant protein production. For the first time, we demonstrated the production of the recombinant protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent assay (iELISA) and the latex agglutination test (LAT).Results Western blotting using known positive serum samples from goats identified a single antigen at the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity, with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using LAT.Conclusion Our findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera

scholarly journals Development and Evaluation of Recombinant GRA8 Protein for the Serodiagnosis of Toxoplasma Gondii Infection in Goats

2020 ◽  
Author(s):  
Charoonluk Jirapattharasate ◽  
Ruenruetai Udonsom ◽  
Apichai Prachasuphap ◽  
Kodcharad Jongpitisub ◽  
Panadda Dhepakson
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Protein ◽  
Sensitivity And Specificity ◽  
Western Blotting ◽  
Sodium Dodecyl ◽  
Laboratory Animals ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Field Samples ◽  
Specific Igg

Abstract Background The development of sensitive and specific methods for detecting Toxoplasma gondii infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently, various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in recombinant protein production. For the first time, we demonstrated the production of the recombinant protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent assay (iELISA) and the latex agglutination test (LAT).Results Western blotting using known positive serum samples from goats identified a single antigen at the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity, with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using LAT.Conclusion Our findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera.

scholarly journals Development of a Specific and Sensitive Enzyme-Linked Immunosorbent Assay as anIn VitroDiagnostic Tool for Detection ofBartonella henselaeAntibodies in Human Serum

2018 ◽  
Vol 56 (12) ◽  
Author(s):  
Markus Jost ◽  
Andreas Latz ◽  
Wibke Ballhorn ◽  
Volkhard A. J. Kempf
Keyword(s):  
Bartonella Henselae ◽  
Insoluble Fraction ◽  
Exchange Chromatography ◽  
Medical Community ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Content Type ◽  
Production Platform ◽  
High Throughput Method ◽  
Human Sera

ABSTRACTBartonella henselaecauses cat scratch disease and several other clinical entities. Infections withB. henselaeare frequently occurring; however, the infection is only rarely diagnosed, mainly due to a lack of knowledge in the medical community. Microscopic immunofluorescence assays (IFA) are widely used for the serodiagnosis ofB. henselaeinfections but are laborious and time-consuming, and interpretation is subjective. An easy and reliable method for the serological diagnosis ofB. henselaeinfections is needed to overcome the shortcomings of the current IFA. Here, we report the development of an ELISA detecting human anti-B. henselaeantibodies from serum samples. By separating the water-insoluble fraction ofB. henselaeHouston-1 via ion-exchange chromatography, 16 subfractions were generated and tested for immunoreactivity via line blotting. One particular fraction (fraction 24) was selected and spotted on ELISA plates using an industrial production platform. By use of well-characterized human sera from the strictly quality-controlled serum library of the German National Consiliary Laboratory forBartonellainfections, the sensitivity of this ELISA was 100% for PCR-proven infections and 76% for clinically suspected infections at a specificity of 93%. This ELISA is therefore a reliable high-throughput method allowing the serodiagnosis ofB. henselaeinfections.

scholarly journals Evaluation of a NovelAspergillusAntigen Enzyme-Linked Immunosorbent Assay

2019 ◽  
Vol 57 (7) ◽  
Author(s):  
Karl Dichtl ◽  
Ulrich Seybold ◽  
Steffen Ormanns ◽  
Heidi Horns ◽  
Johannes Wagener
Keyword(s):  
Sensitivity And Specificity ◽  
Immunosorbent Assay ◽  
Fungal Infections ◽  
Time Frame ◽  
Enzyme Linked Immunosorbent Assay ◽  
The Novel ◽  
Serum Samples ◽  
European Organization ◽  
Content Type ◽  
Patients At Risk

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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