Toxoplasma gondii Recombinant antigen AMA1: Diagnostic Utility of Protein Fragments for the Detection of IgG and IgM Antibodies

Toxoplasma gondii is an important zoonotic protozoan that infects a wide variety of vertebrates as intermediate hosts. For this reason, the diagnosis of this disease is very important and requires continuous improvement. One possibility is to use recombinant antigens in serological tests. Apical membrane antigen 1 (AMA1), a protein located in specific secretory organelles (micronemes) of T. gondii, is very interesting in regard to its potential diagnostic utility. In the present study, we attempted to identify a fragment of the AMA1 protein with a high sensitivity and specificity for the serological diagnosis of human toxoplasmosis. The full-length AMA1 and two different fragments (AMA1N and AMA1C) were produced using an Escherichia coli expression system. After purification by metal affinity chromatography, recombinant proteins were tested for their utility as antigens in enzyme-linked immunosorbent assays (ELISAs) for the detection of IgG and IgM anti-T. gondii antibodies in human and mouse immune sera. Our data demonstrate that the full-length AMA1 recombinant antigen (corresponding to amino acid residues 67–569 of the native protein) has a better diagnostic potential than its N- or C-terminal fragments. This recombinant protein strongly interacts with specific anti-T. gondii IgG (99.4%) and IgM (80.0%) antibodies, and may be used for developing new tools for diagnostics of toxoplasmosis.

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Fluorescent bead-based serological detection of Toxoplasma gondii infection in chickens

10.21203/rs.3.rs-34121/v2 ◽

2020 ◽

Author(s):

Benedikt T. Fabian ◽

Fatima Hedar ◽

Martin Koethe ◽

Berit Bangoura ◽

Pavlo Maksimov ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Small Ruminants ◽

High Sensitivity ◽

Negative Control ◽

Immunofluorescence Assay ◽

Farm Animals ◽

Serological Tests ◽

Modified Agglutination Test ◽

Magnetic Capture ◽

Luminex Technology

Abstract Background: Free-ranging chickens are often infected with Toxoplasma gondii. Their infection indicates environmental contamination with T. gondii. The detection of infected birds relies primarily on serological assays. Methods: Here, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the specific and sensitive detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1bio) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. Serum antibodies were detected by a fluorophore-coupled secondary antibody. Beads of differing color codes were conjugated with anti-chicken IgY or chicken serum albumin and served for each sample as an internal positive or negative control, respectively. The assay was validated with sera from experimentally and naturally infected chickens. The results were compared to those from reference methods, including other serological tests and bioassay in mice.Results: In experimentally infected chickens, the vast majority (98.5%, n=65/66) of inoculated birds tested seropositive in the BBMA. This included all chickens positive by magnetic-capture PCR (100%, n=45/45). Most, but not all inoculated and TgSAG1bio-BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs (TgSAG1-ELISASL, n=61/65; or TgSAG1-ELISASH, n=60/65), or positive in an immunofluorescence assay (IFAT, n=64/65)) and in a modified agglutination test (MAT, n=61/65). All non-inoculated control animals (n=28/28, 100%) tested negative. In naturally exposed chickens, the TgSAG1bio-BBMA showed a high sensitivity (98.5%; 95% Confidence Interval: 90.7-99.9%) and specificity (100%; 85.0-100%) relative to a reference standard established using ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in bioassay or by PCR tested positive in the TgSAG1bio-BBMA (93.5%; 77.1-98.9%), while all bioassay- or PCR-negative chickens remained negative (100%; 85.0-100%).Conclusions: The TgSAG1bio-BBMA represents a suitable method for the detection of T. gondii infections in chickens with high sensitivity and specificity, which is comparable or even superior to other tests. Since assays based on this methodology allow for the simultaneous analysis of a single biological sample with respect to multiple analytes, the described assay may represent one component in future multiplex assays for broad serological monitoring of poultry and other farm animals, including pigs or small ruminants, for various pathogens.

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Diagnostic-test evaluation of immunoassays for anti-Toxoplasma gondii IgG antibodies in a random sample of Mexican population

The Journal of Infection in Developing Countries ◽

10.3855/jidc.3858 ◽

2014 ◽

Vol 8 (05) ◽

pp. 642-647 ◽

Cited By ~ 7

Author(s):

Heriberto Caballero-Ortega ◽

Rocío Castillo-Cruz ◽

Sandra Murieta ◽

Luz Belinda Ortíz-Alegría ◽

Esther Calderón-Segura ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Western Blot ◽

Latin American ◽

High Sensitivity ◽

Reference Standards ◽

Igg Antibodies ◽

Serum Samples ◽

Serological Tests ◽

Western Blots ◽

Open Population

Introduction: There are few articles on evaluation of Toxoplasma gondii serological tests. Besides, commercially available tests are not always useful and are expensive for studies in open population. The aim of this study was to evaluate in-house ELISA and western blot for IgG antibodies in a representative sample of people living in Mexico. Methodology: Three hundred and five serum samples were randomly selected from two national seroepidemiological survey banks; they were taken from men and women of all ages and from all areas of the country. ELISA cut-off was established using the mean plus three standard deviations of negative samples. Western blots were analysed by two experienced technicians and positivity was established according to the presence of at least three diagnostic bands. A commercial ELISA kit was used as a third test. Two reference standards were built up: one using concordant results of two assays leaving the evaluated test out and the other in which the evaluated test was included (IN) with at least two concordant results to define diagnosis. Results: the lowest values of diagnostic parameters were obtained with the OUT reference standards: in-house ELISA had 96.9% sensitivity, 62.1% specificity, 49.6% PPV, 98.1% NPV and 71.8% accuracy, while western blot presented 81.8%, 89.7%, 84.0%, 88.2% and 86.6% values and the best kappa coefficient (0.72-0.82). Conclusions: The in-house ELISA is useful for screening people of Mexico, due to its high sensitivity, while western blot may be used to confirm diagnosis. These techniques might prove useful in other Latin American countries.

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Use of MAG1 Recombinant Antigen for Diagnosis of Toxoplasma gondii Infection in Humans

Clinical and Vaccine Immunology ◽

10.1128/cvi.00419-06 ◽

2007 ◽

Vol 14 (3) ◽

pp. 220-225 ◽

Cited By ~ 32

Author(s):

Lucyna Holec ◽

Elżbieta Hiszczyńska-Sawicka ◽

Artur Gąsior ◽

Anna Brillowska-Dąbrowska ◽

Józef Kur

Keyword(s):

Toxoplasma Gondii ◽

Diagnostic Tests ◽

Expression System ◽

Recombinant Antigen ◽

Acute Stage ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Acute Toxoplasmosis ◽

Toxoplasma Gondii Infection ◽

Potential Use

ABSTRACT This paper describes the cloning, purification, and serological applications of matrix antigen MAG1 of Toxoplasma gondii. The expression system used allows the production of a large amount of T. gondii recombinant protein, which was assessed for its potential use in an enzyme-linked immunosorbent assay (ELISA) for detection of T. gondii infection in humans. Serum samples from 117 patients with different stages of infection, along with 10 serum samples from seronegative patients obtained for routine diagnostic tests, were used. The results were compared with those of an ELISA that uses a native T. gondii antigen extract. The MAG1 antigen detected antibodies more frequently from the acute stage (97.3%) than from the chronic stage (7.5%) of toxoplasmosis. Hence, this antigen may be used as a tool for detection of T. gondii immunoglobulin G antibodies in persons with acute toxoplasmosis.

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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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Inexpensive Designer Antigen for Anti-HIV Antibody Detection with High Sensitivity and Specificity

Clinical and Vaccine Immunology ◽

10.1128/cvi.00283-09 ◽

2010 ◽

Vol 17 (3) ◽

pp. 335-341 ◽

Cited By ~ 4

Author(s):

Sheikh M. Talha ◽

Teppo Salminen ◽

Deepti A. Chugh ◽

Sathyamangalam Swaminathan ◽

Tero Soukka ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Expression System ◽

High Sensitivity ◽

Antibody Detection ◽

Human Antibody ◽

Time Resolved ◽

Reading Frame ◽

Diagnostic Potential ◽

Hiv Antibodies ◽

Anti Hiv

ABSTRACT A novel recombinant multiepitope protein (MEP) has been designed that consists of four linear, immunodominant, and phylogenetically conserved epitopes, taken from human immunodeficiency virus (HIV)-encoded antigens that are used in many third-generation immunoassay kits. This HIV-MEP has been evaluated for its diagnostic potential in the detection of anti-HIV antibodies in human sera. A synthetic MEP gene encoding these epitopes, joined by flexible peptide linkers in a single open reading frame, was designed and overexpressed in Escherichia coli. The recombinant HIV-MEP was purified using a single affinity step, yielding >20 mg pure protein/liter culture, and used as the coating antigen in an in-house immunoassay. Bound anti-HIV antibodies were detected by highly sensitive time-resolved fluorometry, using europium(III) chelate-labeled anti-human antibody. The sensitivity and specificity of the HIV-MEP were evaluated using Boston Biomedica worldwide HIV performance, HIV seroconversion, and viral coinfection panels and were found to be comparable with those of commercially available anti-HIV enzyme immunoassay (EIA) kits. The careful choice of epitopes, high epitope density, and an E. coli-based expression system, coupled with a simple purification protocol and the use of europium(III) chelate-labeled tracer, provide the capability for the development of an inexpensive diagnostic test with high degrees of sensitivity and specificity.

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Fluorescent bead-based serological detection of Toxoplasma gondii infection in chickens

10.21203/rs.3.rs-34121/v3 ◽

2020 ◽

Author(s):

Benedikt T. Fabian ◽

Fatima Hedar ◽

Martin Koethe ◽

Berit Bangoura ◽

Pavlo Maksimov ◽

...

Keyword(s):

Toxoplasma Gondii ◽

High Sensitivity ◽

Negative Control ◽

Immunofluorescence Assay ◽

Farm Animals ◽

Serological Tests ◽

Modified Agglutination Test ◽

Magnetic Capture ◽

Luminex Technology ◽

Toxoplasma Gondii Infection

Abstract Background: Free-ranging chickens are often infected with Toxoplasma gondii and seroconvert upon infection. This indicates environmental contamination with T. gondii. Methods: Here, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1bio) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. Serum antibodies were detected by a fluorophore-coupled secondary antibody. Beads of differing color codes were conjugated with anti-chicken IgY or chicken serum albumin and served for each sample as an internal positive or negative control, respectively. The assay was validated with sera from experimentally and naturally infected chickens. The results were compared to those from reference methods, including other serological tests, PCRs and bioassay in mice.Results: In experimentally infected chickens, the vast majority (98.5%, n = 65/66) of birds tested seropositive in the BBMA. This included all chickens positive by magnetic-capture PCR (100%, n = 45/45). Most, but not all inoculated and TgSAG1bio-BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs (TgSAG1-ELISASL, n = 61/65; or TgSAG1-ELISASH, n = 60/65), or positive in an immunofluorescence assay (IFAT, n = 64/65)) and in a modified agglutination test (MAT, n = 61/65). All non-inoculated control animals (n = 28/28, 100%) tested negative. In naturally exposed chickens, the TgSAG1bio-BBMA showed a high sensitivity (98.5%; 95% confidence interval, CI: 90.7–99.9%) and specificity (100%; 95% CI: 85.0–100%) relative to a reference standard established using ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in bioassay or by PCR tested positive in the TgSAG1bio-BBMA (93.5%; 95% CI: 77.1–98.9%), while all bioassay- or PCR-negative chickens remained negative (100%; 95% CI: 85.0–100%).Conclusions: The TgSAG1bio-BBMA represents a suitable method for the detection of T. gondii infections in chickens with high sensitivity and specificity, which is comparable or even superior to other tests. Since assays based on this methodology allow for the simultaneous analysis of a single biological sample with respect to multiple analytes, the described assay may represent a component in future multiplex assays for broad serological monitoring of poultry and other farm animals for various pathogens.

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Fluorescent bead-based serological detection of Toxoplasma gondii infection in chickens

10.21203/rs.3.rs-34121/v1 ◽

2020 ◽

Author(s):

Benedikt T. Fabian ◽

Fatima Hedar ◽

Martin Koethe ◽

Berit Bangoura ◽

Pavlo Maksimov ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Small Ruminants ◽

High Sensitivity ◽

Negative Control ◽

Immunofluorescence Assay ◽

Farm Animals ◽

Mouse Bioassay ◽

Serological Tests ◽

Magnetic Capture ◽

Luminex Technology

Abstract Background: Especially free-ranging chickens are frequently exposed to Toxoplasma gondii. They are sensitive indicators for environmental contamination with T. gondii oocysts. The detection of infected birds relies primarily on serological assays. Methods: Here, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the specific and sensitive detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1bio) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. Specific serum antibodies were detected by a fluorophore-coupled secondary antibody. Beads of differing color codes were conjugated with anti-chicken IgY or chicken serum albumin and served for each individual sample as an internal positive or negative control, respectively. The assay was validated with sera from experimentally and naturally infected chickens. The results were compared to those from reference methods, including other serological tests and bioassay in mice.Results: In experimentally infected chickens, all chickens were positive by magnetic-capture PCR (100%, n=45/45) and the vast majority (98.5%, n=65/66) of inoculated birds tested seropositive in the BBMA. Most, but not all inoculated and TgSAG1bio-BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs, an immunofluorescence assay (IFAT) and in a modified agglutination test (MAT). All non-inoculated control animals (n=28) tested negative. In naturally exposed chickens, the TgSAG1bio-BBMA showed a high sensitivity (98.5%; 95% Confidence Interval: 90.7-99.9%) and specificity (100%; 85.0-100%) relative to a reference standard established using results obtained with ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in the mouse bioassay or by PCR tested positive in the TgSAG1bio-BBMA (93.5%, 77.1-98.9%), while all mouse bioassay- or PCR-negative chickens remained negative (100%, 85.0-100%).Conclusions: The TgSAG1bio-BBMA represents a suitable method for the detection of T. gondii infections in chickens with high sensitivity and specificity, which is comparable or even superior to other tests. Since assays based on this methodology allow for the simultaneous analysis of a single biological sample with respect to multiple analytes, the described assay may represent one component in future multiplex assays for broad serological monitoring of poultry and other farm animals, including pigs or small ruminants, for various pathogens.

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TO SEPARATELY ASSESS THE DIAGNOSTIC POTENTIAL OF TVS & MRI IN THE TERMS OF SENSITIVITY AND SPECIFICITY BY CORRELATING THE IMAGING FINDINGS WITH HISTOPATHOLOGY

International Journal of Medical and Biomedical Studies ◽

10.32553/ijmbs.v4i6.1209 ◽

2020 ◽

Vol 4 (6) ◽

Author(s):

Suraj Mathur

Keyword(s):

Transvaginal Ultrasound ◽

High Sensitivity ◽

Imaging Evaluation ◽

Medical College ◽

Conventional Mri ◽

Diagnostic Potential ◽

Adc Mapping ◽

Malignant Lesions ◽

Sensitivity Specificity

This prospective study was done in the Department of Radio diagnosis Govt. Medical College, Kozhikode. A total of 65 patients who were referred to our department with clinical suspicion of endometrial lesions and incidentally detected endometrial lesions on ultrasonography underwent transvaginal ultrasound and subsequent Imaging evaluation of pelvis MRI has very high sensitivity (95%) and specificity (98%) and is almost as accurate (97%) as histopathology in differentiating benign from malignant lesions. Addition of DWI with ADC mapping to conventional MRI increases its accuracy even more. However there is inherent limitation to MRI in detecting carcinoma in situ and micrometastasis. Keywords: TVS, MRI, Sensitivity, Specificity, Histopathology.

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Analytical Validation and Clinical Application of Rapid Serological Tests for SARS-CoV-2 Suitable for Large-Scale Screening

Diagnostics ◽

10.3390/diagnostics11050869 ◽

2021 ◽

Vol 11 (5) ◽

pp. 869

Author(s):

Amedeo De Nicolò ◽

Valeria Avataneo ◽

Jessica Cusato ◽

Alice Palermiti ◽

Jacopo Mula ◽

...

Keyword(s):

Early Diagnosis ◽

Real Time Pcr ◽

Large Scale ◽

High Sensitivity ◽

Analytical Validation ◽

Pcr Assay ◽

Serological Tests ◽

Flow Test ◽

Lateral Flow Test ◽

Large Scale Screening

Recently, large-scale screening for COVID-19 has presented a major challenge, limiting timely countermeasures. Therefore, the application of suitable rapid serological tests could provide useful information, however, little evidence regarding their robustness is currently available. In this work, we evaluated and compared the analytical performance of a rapid lateral-flow test (LFA) and a fast semiquantitative fluorescent immunoassay (FIA) for anti-nucleocapsid (anti-NC) antibodies, with the reverse transcriptase real-time PCR assay as the reference. In 222 patients, LFA showed poor sensitivity (55.9%) within two weeks from PCR, while later testing was more reliable (sensitivity of 85.7% and specificity of 93.1%). Moreover, in a subset of 100 patients, FIA showed high sensitivity (89.1%) and specificity (94.1%) after two weeks from PCR. The coupled application for the screening of 183 patients showed satisfactory concordance (K = 0.858). In conclusion, rapid serological tests were largely not useful for early diagnosis, but they showed good performance in later stages of infection. These could be useful for back-tracing and/or to identify potentially immune subjects.

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Neuroendocrine Neoplasms: Identification of Novel Metabolic Circuits of Potential Diagnostic Utility

Cancers ◽

10.3390/cancers13030374 ◽

2021 ◽

Vol 13 (3) ◽

pp. 374

Author(s):

Beatriz Jiménez ◽

Mei Ran Abellona U ◽

Panagiotis Drymousis ◽

Michael Kyriakides ◽

Ashley K. Clift ◽

...

Keyword(s):

1H Nmr ◽

Cancer Metabolism ◽

Proton Nuclear Magnetic Resonance ◽

Neuroendocrine Neoplasms ◽

Resonance Spectroscopy ◽

Diagnostic Utility ◽

Signalling Molecules ◽

Prognostic Accuracy ◽

Diagnostic Potential ◽

Healthy Control

The incidence of neuroendocrine neoplasms (NEN) is increasing, but established biomarkers have poor diagnostic and prognostic accuracy. Here, we aim to define the systemic metabolic consequences of NEN and to establish the diagnostic utility of proton nuclear magnetic resonance spectroscopy (1H-NMR) for NEN in a prospective cohort of patients through a single-centre, prospective controlled observational study. Urine samples of 34 treatment-naïve NEN patients (median age: 59.3 years, range: 36–85): 18 had pancreatic (Pan) NEN, of which seven were functioning; 16 had small bowel (SB) NEN; 20 age- and sex-matched healthy control individuals were analysed using a 600 MHz Bruker 1H-NMR spectrometer. Orthogonal partial-least-squares-discriminant analysis models were able to discriminate both PanNEN and SBNEN patients from healthy control (Healthy vs. PanNEN: AUC = 0.90, Healthy vs. SBNEN: AUC = 0.90). Secondary metabolites of tryptophan, such as trigonelline and a niacin-related metabolite were also identified to be universally decreased in NEN patients, while upstream metabolites, such as kynurenine, were elevated in SBNEN. Hippurate, a gut-derived metabolite, was reduced in all patients, whereas other gut microbial co-metabolites, trimethylamine-N-oxide, 4-hydroxyphenylacetate and phenylacetylglutamine, were elevated in those with SBNEN. These findings suggest the existence of a new systems-based neuroendocrine circuit, regulated in part by cancer metabolism, neuroendocrine signalling molecules and gut microbial co-metabolism. Metabonomic profiling of NEN has diagnostic potential and could be used for discovering biomarkers for these tumours. These preliminary data require confirmation in a larger cohort.

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