scholarly journals Heterologous Expression, Purification, and Immunological Reactivity of a Recombinant HSP60 from Paracoccidioides brasiliensis

2002 ◽  
Vol 9 (2) ◽  
pp. 374-377 ◽  
Author(s):  
Daniela A. Cunha ◽  
Roseli M. Zancopé-Oliveira ◽  
M. Sueli ◽  
S. Felipe ◽  
Silvia M. Salem-Izacc ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Paracoccidioides Brasiliensis ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Antibody Responses ◽  
Healthy Individuals ◽  
Immunological Reactivity ◽  
Serum Samples ◽  
Recombinant Antigens

ABSTRACT The complete coding cDNA of HSP60 from Paracoccidioides brasiliensis was overexpressed in an Escherichia coli host to produce high levels of recombinant protein. The protein was purified by affinity chromatography. A total of 169 human serum samples were tested for reactivity by Western blot analysis with the purified HSP60 recombinant protein. Immunoblots indicated that the recombinant P. brasiliensis HSP60 was recognized by antibodies in 72 of 75 sera from paracoccidioidomycosis patients. No cross-reactivity was detected with individual sera from patients with aspergillosis, sporotrichosis, cryptococcosis, and tuberculosis. Reactivity to HSP60 was observed in sera from 9.52% of control healthy individuals and 11.5% of patients with histoplasmosis. The high sensitivity and specificity (97.3 and 92.5%, respectively) for HSP60 suggested that the recombinant protein can be used singly or in association with other recombinant antigens to detect antibody responses in P. brasiliensis-infected patients.

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

1999 ◽  
Vol 37 (5) ◽  
pp. 1554-1560 ◽  
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.

scholarly journals Comparison of Ehrlichia chaffeensisRecombinant Proteins for Serologic Diagnosis of Human Monocytotropic Ehrlichiosis

1999 ◽  
Vol 37 (8) ◽  
pp. 2568-2575 ◽  
Author(s):  
Xue-Jie Yu ◽  
Patricia A. Crocquet-Valdes ◽  
Louis C. Cullman ◽  
Vsevolod L. Popov ◽  
David H. Walker
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Gram Negative Bacteria ◽  
Iron Binding ◽  
Serum Samples ◽  
Gram Negative ◽  
Recombinant Antigens ◽  
Genes Encoding ◽  
Iron Binding Protein ◽  
Positive Results

Diagnosis of human monocytotropic ehrlichiosis (HME) generally depends on serology that detects the antibody response to immunodominant proteins of Ehrlichia chaffeensis. Protein immunoblotting was used to evaluate the reaction of the antibodies in patients’ sera with the recombinant E. chaffeensis 120- and 28-kDa proteins as well as the 106- and the 37-kDa proteins. The cloning of the genes encoding the latter two proteins is described in this report. Immunoelectron microscopy demonstrated that the 106-kDa protein is located at the surfaces of ehrlichiae and on the intramorular fibrillar structures associated with E. chaffeensis. The 37-kDa protein is homologous to the iron-binding protein of gram-negative bacteria. Forty-two serum samples from patients who were suspected to have HME were tested by immunofluorescence (IFA) using E. chaffeensis antigen and by protein immunoblotting using recombinant E. chaffeensisproteins expressed in Escherichia coli. Thirty-two serum samples contained IFA antibodies at a titer of 1:64 or greater. The correlation of IFA and recombinant protein immunoblotting was 100% for the 120-kDa protein, 41% for the 28-kDa protein, 9.4% for the 106-kDa protein, and 0% for the 37-kDa protein. None of the recombinant antigens yielded false-positive results. All the sera reactive with the recombinant 28- or the 106-kDa proteins also reacted with the recombinant 120-kDa protein.

scholarly journals EVALUATION OF ELEVEN IMMUNOCHROMATOGRAPHIC ASSAYS FOR SARS-CoV-2 DETECTION: INVESTIGATING DENGUE CROSS-REACTION

2020 ◽  
Author(s):  
Beatriz Araujo Oliveira ◽  
Lea Campos de Oliveira ◽  
Franciane Mendes de Oliveira ◽  
Geovana Maria Pereira ◽  
Regina Maia de Souza ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
High Sensitivity ◽  
Diagnostic Techniques ◽  
Cross Reactivity ◽  
Cross Reaction ◽  
List Type ◽  
Rt Pcr ◽  
Serum Samples ◽  
Igm Antibodies ◽  
Good Agreement

AbstractBackgroundCOVID-19 disease (Coronavirus disease 2019) caused by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) is widespread worldwide, affecting more than 11 million people globally (July 6th, 2020). Diagnostic techniques have been studied in order to contain the pandemic. Immunochromatographic (IC) assays are feasible and low cost alternative for monitoring the spread of COVID-19 in the population.MethodsHere we evaluate the sensitivity and specificity of eleven different immunochromatographic tests in 98 serum samples from confirmed cases of COVID-19 through RT-PCR and 100 negative serum samples from blood donors collected in February 2019. Considering the endemic situation of Dengue in Brazil, we also evaluated the cross-reactivity with Dengue using 20 serum samples from patients with confirmed diagnosis for Dengue collected in early 2019 through four different tests.ResultsOur results demonstrated agreement between immunochromatographic assays and RT-PCR, especially after 10 days since the onset of symptoms. The evaluation of IgG and IgM antibodies combined demonstrated a strong level of agreement (0.85) of IC assays and RT-PCR. It was observed cross-reactivity between Dengue and COVID-19 using four different IC assays for COVID-19 diagnosis. The specificity of IC assays to detected COVID-19 IgM antibodies using Dengue serum samples varied from 80% to 85%; the specificity of IgG detection was 100% and total antibody was 95%.ConclusionsWe found high sensitivity, specificity and good agreement of IC assays, especially after 10 days onset of symptoms. However, we detected cross-reactivity between Dengue and COVID-19 mainly with IgM antibodies demonstrating the need for better studies about diagnostic techniques for these diseases.HighlightsImmunochromatographic assays demonstrated high sensitivity and specificity and good agreement with the gold-standard RT-PCR;Increase in sensitivity and specificity of assays using samples collected after the 10th day of symptoms;Cross-reaction with Dengue serology in evaluation of IgM.

A rapid, sensitive enzyme-linked immunoassay for human thyrotropin.

1985 ◽  
Vol 31 (7) ◽  
pp. 1131-1134 ◽  
Author(s):  
Y C Tseng ◽  
K D Burman ◽  
J R Baker ◽  
L Wartofsky
Keyword(s):  
Color Change ◽  
Clinical Laboratory ◽  
High Sensitivity ◽  
Normal Subjects ◽  
Turnaround Time ◽  
Cross Reactivity ◽  
Serum Samples ◽  
Assay Sensitivity ◽  
Nitrophenyl Phosphate ◽  
Set Up

Abstract In this enzyme-linked immunoassay for human thyrotropin (TSH) in unextracted serum we use 96-well immunoenzymometric assay plates, first coated with polyclonal antibody to TSH, then incubated with the serum samples and reacted with mouse monoclonal antibody to human TSH. After incubation with alkaline phosphatase-labeled antibody against mouse IgG, disodium p-nitrophenyl phosphate is added and the color change is measured spectrophotometrically. Assay sensitivity is 0.1 milli-int. unit/L. Cross reactivity with lutropin, follitropin, or choriogonadotropin was negligible. TSH concentrations ranged from 0.4 to 4.1 milli-int. units/L in 43 normal subjects (mean 2.0, SD 1.0), and were uniformly less than 0.3 milli-int. unit/L in 23 patients with hyperthyroidism. Features which make this assay advantageous to the clinical laboratory include ease of set-up, ability to assay many samples at a time, high sensitivity, rapid turnaround time (8 h), and absence of requirements for radioactive materials.

Cross-reactivity and avidity of antibody responses induced in humans by the oral inactivated multivalent enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX

Vaccine ◽  
2017 ◽  
Vol 35 (32) ◽  
pp. 3966-3973 ◽  
Author(s):  
Susannah Leach ◽  
Anna Lundgren ◽  
Nils Carlin ◽  
Madeleine Löfstrand ◽  
Ann-Mari Svennerholm
Keyword(s):  
Escherichia Coli ◽  
Cross Reactivity ◽  
Antibody Responses ◽  
Enterotoxigenic Escherichia Coli

scholarly journals Development and Evaluation of a Latex Agglutination Test for the Serodiagnosis of Paracoccidioidomycosis

2011 ◽  
Vol 18 (4) ◽  
pp. 604-608 ◽  
Author(s):  
Fabíola Silveira-Gomes ◽  
Dayse Nogueira Sarmento ◽  
Thifany Mendes Pinto ◽  
Rosiane Ferreira Pimentel ◽  
Lívia Barreto Nepomuceno ◽  
...  
Keyword(s):  
Latex Particle ◽  
Paracoccidioides Brasiliensis ◽  
Cross Reactivity ◽  
Latex Agglutination ◽  
Dimorphic Fungus ◽  
Double Immunodiffusion ◽  
Serum Samples ◽  
Content Type ◽  
Healthy Donors ◽  
Low Costs

ABSTRACTParacoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. It is caused by the dimorphic fungusParacoccidioides brasiliensis. The immunodiffusion (ID) test is one of the most widely used techniques for PCM serologic diagnosis due to the simplicity and low costs of its execution. However, it requires trained and qualified people to execute it. The purpose of this study was to evaluate a latex particle agglutination (LA) test for the detection of anti-P. brasiliensisantibodies by using pooled crude exoantigens from the fungus. Fifty-one serum samples obtained from patients with PCM were tested. Positivity was observed in 84% (43/51) of these patients, and the agglutination patterns varied from small clumps with a cloudy background to large clumps with a clear background. The antibody titer reactivity ranged from 1:2 to 1:64. Cross-reactivity was observed in sera from patients with aspergillosis, histoplasmosis, and nonfungal disease. Serum samples obtained from healthy donors were not reactive. The sensitivity and specificity of the LA test were 84% and 81%, respectively. When comparing the LA test with the double-immunodiffusion test, we found an agreement of 92%. Further work is needed to improve the performance of the LA assay before it can be proposed as a reliable diagnostic tool, mainly in laboratories with little infrastructure.

scholarly journals Extracellular Vesicle-Based Detection of Pancreatic Cancer

2021 ◽  
Vol 9 ◽  
Author(s):  
Yesim Verel-Yilmaz ◽  
Juan Pablo Fernández ◽  
Agnes Schäfer ◽  
Sheila Nevermann ◽  
Lena Cook ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Roc Curves ◽  
Serum Levels ◽  
Extracellular Vesicle ◽  
Ductal Adenocarcinoma ◽  
Rank Test ◽  
Healthy Controls ◽  
Precursor Lesions ◽  
Healthy Individuals ◽  
Serum Samples

Due to a grim prognosis, there is an urgent need to detect pancreatic ductal adenocarcinoma (PDAC) prior to metastasis. However, reliable diagnostic imaging methods or biomarkers for PDAC or its precursor lesions are still scarce. ADAM8, a metalloprotease-disintegrin, is highly expressed in PDAC tissue and negatively correlates with patient survival. The aim of our study was to determine the ability of ADAM8-positive extracellular vesicles (EVs) and cargo microRNAs (miRNAs) to discriminate precursor lesions or PDAC from healthy controls. In order to investigate enrichment of ADAM8 on EVs, these were isolated from serum of patients with PDAC (n = 52), precursor lesions (n = 7) and healthy individuals (n = 20). Nanoparticle Tracking Analysis and electron microscopy indicated successful preparation of EVs that were analyzed for ADAM8 by FACS. Additionally, EV cargo analyses of miRNAs from the same serum samples revealed the presence of miR-720 and miR-451 by qPCR and was validated in 20 additional PDAC samples. Statistical analyses included Wilcoxon rank test and ROC curves. FACS analysis detected significant enrichment of ADAM8 in EVs from patients with PDAC or precursor lesions compared to healthy individuals (p = 0.0005). ADAM8-dependent co-variates, miR-451 and miR-720 were also diagnostic, as patients with PDAC had significantly higher serum levels of miR-451 and lower serum levels of miR-720 than healthy controls and reached high sensitivity and specificity (AUC = 0.93 and 1.00, respectively) to discriminate PDAC from healthy control. Thus, detection of ADAM8-positive EVs and related cargo miR-720 and miR-451 may constitute a specific biomarker set for screening individuals at risk for PDAC.

scholarly journals Specific Serodiagnosis of Canine Visceral Leishmaniasis Using Leishmania Species Ribosomal Protein Extracts

2009 ◽  
Vol 16 (12) ◽  
pp. 1774-1780 ◽  
Author(s):  
Eduardo A. F. Coelho ◽  
Laura Ramírez ◽  
Mariana A. F. Costa ◽  
Vinicio T. S. Coelho ◽  
Vivian T. Martins ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Ribosomal Proteins ◽  
High Sensitivity ◽  
Leishmania Species ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Canine Visceral Leishmaniasis ◽  
Leishmania Chagasi ◽  
Serum Samples ◽  
Potential Tool

ABSTRACT In the present work, we have analyzed the antigenicity of Leishmania species ribosomal proteins (LRPs). To accomplish this, Leishmania infantum ribosomes were biochemically purified from promastigote cytosolic extracts, and their reactivities were analyzed by using the sera from dogs naturally infected with L. infantum. Since antibodies reacting against different ribosomal proteins were observed in all the serum samples obtained from dogs with symptomatic visceral leishmaniasis tested, we have analyzed the potential usefulness of the LRP extracts in the development of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of canine visceral leishmaniasis (CVL) in an area of Brazil where visceral leishmaniasis is endemic due to infection by Leishmania chagasi. A comparative ELISA with crude soluble Leishmania chagasi antigen (SLA) and L. infantum LRPs was performed. LRP- and SLA-based ELISAs gave similar sensitivities for the diagnosis of symptomatic CVL, but the LRP extract provided a very high sensitivity for the detection of oligosymptomatic and asymptomatic dogs. In addition, an LRP-based ELISA showed a higher specificity when the sera from dogs harboring other infections were included in the analysis. The LRP antigen displayed no cross-reactivity with sera from dogs that had any of the other diseases tested, notably, Chagas' disease. Our findings suggest that LRPs are a potential tool for the diagnosis of CVL and will be particularly useful for the diagnosis of asymptomatic CVL.

scholarly journals Expression in Bacteria of the Gene Encoding the gp43 Antigen of Paracoccidioides brasiliensis: Immunological Reactivity of the Recombinant Fusion Proteins

2002 ◽  
Vol 9 (6) ◽  
pp. 1200-1204 ◽  
Author(s):  
Susana N. Diniz ◽  
Kátia C. Carvalho ◽  
Patrícia S. Cisalpino ◽  
José F. Silveira ◽  
Luiz R. Travassos ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Paracoccidioides Brasiliensis ◽  
Immunological Reactivity ◽  
Gene Encoding ◽  
Recombinant Fusion Proteins ◽  
Gst Fusion Protein

ABSTRACT gp43 is the major diagnostic antigen of Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis (PCM) in humans. In the present study, cDNA of the gp43 gene (PbGP43) was obtained by reverse transcriptase PCR, inserted into a pGEX vector in frame with the glutathione S-transferase (GST) gene, and expressed in Escherichia coli as inclusion bodies. Immunoblotting showed that all sera from patients with chronic pulmonary and acute lymphatic forms of PCM reacted with the recombinant fusion protein of the mature gp43 (381 amino acids). Reactivity with fusion proteins containing subfragments of the N-terminal, internal, or C-terminal regions occurred eventually, and the C-terminal region was the most antigenic. Lack of reactivity with the subfragments may be due to the conformational nature of the gp43 epitopes. Sera from patients with aspergillosis, candidiasis, and histoplasmosis did not react with the gp43-GST fusion protein. Our results suggest that recombinant gp43 corresponding to the processed antigen can be a useful tool in the diagnosis of PCM.

scholarly journals Species-Specific Antibody Responses to the Recombinant 53-Kilodalton Excretory and Secretory Proteins in Mice Infected with Trichinella spp.

2008 ◽  
Vol 15 (3) ◽  
pp. 468-473 ◽  
Author(s):  
Isao Nagano ◽  
Zhiliang Wu ◽  
Yuzo Takahashi
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Specific Antibody ◽  
Expression System ◽  
Amino Acid Sequences ◽  
Antibody Responses ◽  
Secretory Proteins ◽  
Muscle Larvae ◽  
Species Specific

ABSTRACT The 53-kDa proteins in larval excretory and secretory (E-S) products were expressed from five Trichinella species (T. spiralis, T. britovi, T. nativa, T. pseudospiralis, and T. papuae), using the Escherichia coli expression system, and the antibody responses to the 53-kDa recombinant proteins in mice infected with Trichinella spp. were analyzed by Western blotting. The 53-kDa protein is conserved among the five Trichinella species, with >60% similarity in amino acid sequences. The 53-kDa recombinant proteins of T. spiralis and T. pseudospiralis reacted to sera from mice infected with T. spiralis and T. pseudospiralis at 8 days postinfection (p.i.), respectively. An antibody against the 53-kDa recombinant protein of T. spiralis recognized the 53-kDa protein in the crude extracts from adult worms and 30-day p.i. muscle larvae and E-S products from muscle larvae of T. spiralis but did not recognize any proteins from T. pseudospiralis. The sera from the mice infected with T. spiralis strongly reacted with the 53-kDa recombinant protein of T. spiralis but did not react with the 53-kDa recombinant proteins of T. britovi, T. nativa, T. pseudospiralis, and T. papuae. Similarly, the sera from mice infected with T. britovi, T. nativa, T. pseudospiralis, or T. papuae strongly reacted with the 53-kDa recombinant proteins of T. britovi, T. nativa, T. pseudospiralis, or T. papuae, respectively. These results showed that the 53-kDa recombinant proteins provide early and species-specific antibody responses in mice infected with Trichinella spp.

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