scholarly journals Epidemic Preparedness—Leishmania tarentolae as an Easy-to-Handle Tool to Produce Antigens for Viral Diagnosis: Application to COVID-19

2021 ◽  
Vol 12 ◽  
Author(s):  
Ilaria Varotto-Boccazzi ◽  
Alessandro Manenti ◽  
Francesca Dapporto ◽  
Louise J. Gourlay ◽  
Beatrice Bisaglia ◽  
...  
Keyword(s):  
Rapid Development ◽  
Antibody Detection ◽  
Molecular Diagnostic ◽  
Serological Tests ◽  
Leishmania Tarentolae ◽  
Cell Factories ◽  
Effective System ◽  
Human Sera ◽  
Antiviral Antibody ◽  
Viral Diagnosis

To detect and prevent emerging epidemics, discovery platforms are urgently needed, for the rapid development of diagnostic assays. Molecular diagnostic tests for COVID-19 were developed shortly after the isolation of SARS-CoV-2. However, serological tests based on antiviral antibody detection, revealing previous exposure to the virus, required longer testing phases, due to the need to obtain correctly folded and glycosylated antigens. The delay between the identification of a new virus and the development of reliable serodiagnostic tools limits our readiness to tackle future epidemics. We suggest that the protozoan Leishmania tarentolae can be used as an easy-to-handle microfactory for the rapid production of viral antigens to face emerging epidemics. We engineered L. tarentolae to express the SARS-CoV-2 receptor-binding domain (RBD) and we recorded the ability of the purified RBD antigen to detect SARS-CoV-2 infection in human sera, with a sensitivity and reproducibility comparable to that of a reference antigen produced in human cells. This is the first application of an antigen produced in L. tarentolae for the serodiagnosis of a Coronaviridae infection. On the basis of our results, we propose L. tarentolae as an effective system for viral antigen production, even in countries that lack high-technology cell factories.

scholarly journals Epidemic preparedness - Leishmania tarentolae as an easy-to-handle tool to produce antigens for viral diagnosis: application to COVID-19

2021 ◽  
Author(s):  
Ilaria Varotto-Boccazzi ◽  
Alessandro Manenti ◽  
Francesca Dapporto ◽  
Louise Gourlay ◽  
Beatrice Bisaglia ◽  
...  
Keyword(s):  
Rapid Development ◽  
Antibody Detection ◽  
Molecular Diagnostic ◽  
High Tech ◽  
Serological Tests ◽  
Leishmania Tarentolae ◽  
Cell Factories ◽  
Effective System ◽  
Antiviral Antibody ◽  
Viral Diagnosis

To control future epidemics, discovery platforms are urgently needed, for the rapid development of diagnostic assays. Molecular diagnostic tests for COVID-19 emerged shortly after the isolation of SARS-CoV-2, however, serological tests based on antiviral antibody detection, revealing previous exposure to the virus, required longer developmental phases, due to the need for correctly folded and glycosylated antigens. The delay between the identification of a new virus and the development of reliable serodiagnostic tools limits our readiness for the control of a future epidemic. In this context, we propose the protozoan Leishmania tarentolae as an easy-to-handle micro-factory for the rapid production of viral antigens, to be used at the forefront of emerging epidemics. As a study model, we engineered L. tarentolae to express the SARS-CoV-2 Receptor Binding Domain (RBD) and report the ability of the purified RBD antigen to detect SARS-CoV-2 infection, with a sensitivity and reproducibility comparable to that of a reference antigen produced in human cells. This is the first application of an antigen produced in L. tarentolae for the serodiagnosis of a Coronaviridae infection. Based on our results, we propose L. tarentolae as an effective system for viral antigen production, even in countries that lack high-tech cell factories.

scholarly journals Leishmania tarentolae and Leishmania infantum in humans, dogs and cats in the Pelagie archipelago, southern Italy

2021 ◽  
Vol 15 (9) ◽  
pp. e0009817
Author(s):  
Roberta Iatta ◽  
Jairo Alfonso Mendoza-Roldan ◽  
Maria Stefania Latrofa ◽  
Antonio Cascio ◽  
Emanuele Brianti ◽  
...  
Keyword(s):  
Leishmania Infantum ◽  
Molecular Diagnostic ◽  
Enzyme Linked Immunosorbent Assay ◽  
Molecular Testing ◽  
Molecular Diagnostic Techniques ◽  
Serum Samples ◽  
Serological Tests ◽  
Leishmania Tarentolae ◽  
Human Samples ◽  
Animal Populations

Visceral leishmaniasis (VL) caused by Leishmania infantum is endemic in the Mediterranean basin with most of the infected human patients remaining asymptomatic. Recently, the saurian-associated Leishmania tarentolae was detected in human blood donors and in sheltered dogs. The circulation of L. infantum and L. tarentolae was investigated in humans, dogs and cats living in the Pelagie islands (Sicily, Italy) by multiple serological and molecular testing. Human serum samples (n = 346) were tested to assess the exposure to L. infantum by immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) and to L. tarentolae by IFAT. Meanwhile, sera from dogs (n = 149) and cats (n = 32) were tested for both Leishmania species by IFAT and all blood samples by specific sets of real time-PCR for L. infantum and L. tarentolae. The agreement between serological tests performed for human samples, and between serological and molecular diagnostic techniques for both human and animal samples were also assessed. Overall, 41 human samples (11.8%, 95% CI: 8.9–15.7) were positive to L. infantum (5.2%, 95% CI: 3.3–8.1), L. tarentolae (5.2%, 95% CI: 3.3–8.1) and to both species (1.4%, 95% CI: 0.6–3.3) by serology and/or molecular tests. A good agreement among the serological tests was determined. Both Leishmania spp. were serologically and/or molecularly detected in 39.6% dogs and 43.7% cats. In addition to L. infantum, also L. tarentolae circulates in human and animal populations, raising relevant public health implications. Further studies should investigate the potential beneficial effects of L. tarentolae in the protection against L. infantum infection.

scholarly journals A new ELISA and western blot technique based on recombinant TES antigen and/or larval antigen for the detection of toxocariasis in humans

Parasitology ◽  
2020 ◽  
pp. 1-8
Author(s):  
Marie-Kristin Raulf ◽  
Daniela Jordan ◽  
Herbert Auer ◽  
Jens M. Warnecke ◽  
Bernd Lepenies ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
High Reliability ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Tests ◽  
Western Blots ◽  
Blot Technique ◽  
Human Sera

Abstract Serological antibody detection by enzyme-linked immunosorbent assay (ELISA)- and immunoblot-based methods constitutes the best indicator of human Toxocara infection. Nevertheless, the availability of serological tests, particularly western blots (WB), evaluated for sensitivity and specificity is limited. Therefore, an Anti-Toxocara-ELISA immunoglobulin g (IgG) prototype (Proto-ELISA) and an Anti-Toxocara-Westernblot (IgG) prototype (Proto-WB) were evaluated by testing 541 human sera pre-determined for Toxocara infection by an established in-house Anti-Toxocara-ELISA (IH-ELISA). To evaluate sensitivity and specificity of the newly developed ELISA and WB prototypes, results were compared to IH-ELISA and a commercial WB (Com-WB). Compared to the IH-ELISA, a sensitivity of 93.1% (229/246) and a specificity of 94.6% (279/295) of the Proto-ELISA with a Cohen's κ of 0.88 were obtained. The sensitivity of the Proto-WB was 76.7% (240/313) and specificity was 99.6% (227/228) with a Cohen's κ of 0.73 compared to those of Com-WB. A comparison to the IH-ELISA revealed 91.5% (225/246) sensitivity and 94.6% (279/295) specificity of the Proto-WB with a Cohen's κ of 0.86. Cross-reactivity was observed for some samples positive for Ascaris and Trichinella spp. in the Proto-ELISA, Proto-WB and Com-WB. Overall, the evaluated ELISA and WB prototypes showed high sensitivity and specificity, indicating high reliability of these newly developed tests.

scholarly journals Myasthenia Gravis: Autoantibody Specificities and Their Role in MG Management

2020 ◽  
Vol 11 ◽  
Author(s):  
Konstantinos Lazaridis ◽  
Socrates J. Tzartos
Keyword(s):  
Neuromuscular Junction ◽  
Myasthenia Gravis ◽  
Clinical Manifestations ◽  
Density Lipoprotein ◽  
Disease Diagnosis ◽  
Autoimmune Response ◽  
Response To Treatment ◽  
Antibody Detection ◽  
Serological Tests ◽  
The Past

Myasthenia gravis (MG) is the most common autoimmune disorder affecting the neuromuscular junction, characterized by skeletal muscle weakness and fatigability. It is caused by autoantibodies targeting proteins of the neuromuscular junction; ~85% of MG patients have autoantibodies against the muscle acetylcholine receptor (AChR-MG), whereas about 5% of MG patients have autoantibodies against the muscle specific kinase (MuSK-MG). In the remaining about 10% of patients no autoantibodies can be found with the classical diagnostics for AChR and MuSK antibodies (seronegative MG, SN-MG). Since serological tests are relatively easy and non-invasive for disease diagnosis, the improvement of methods for the detection of known autoantibodies or the discovery of novel autoantibody specificities to diminish SN-MG and to facilitate differential diagnosis of similar diseases, is crucial. Radioimmunoprecipitation assays (RIPA) are the staple for MG antibody detection, but over the past years, using cell-based assays (CBAs) or improved highly sensitive RIPAs, it has been possible to detect autoantibodies in previously SN-MG patients. This led to the identification of more patients with antibodies to the classical antigens AChR and MuSK and to the third MG autoantigen, the low-density lipoprotein receptor-related protein 4 (LRP4), while antibodies against other extracellular or intracellular targets, such as agrin, Kv1.4 potassium channels, collagen Q, titin, the ryanodine receptor and cortactin have been found in some MG patients. Since the autoantigen targeted determines in part the clinical manifestations, prognosis and response to treatment, serological tests are not only indispensable for initial diagnosis, but also for monitoring treatment efficacy. Importantly, knowing the autoantibody profile of MG patients could allow for more efficient personalized therapeutic approaches. Significant progress has been made over the past years toward the development of antigen-specific therapies, targeting only the specific immune cells or autoantibodies involved in the autoimmune response. In this review, we will present the progress made toward the development of novel sensitive autoantibody detection assays, the identification of new MG autoantigens, and the implications for improved antigen-specific therapeutics. These advancements increase our understanding of MG pathology and improve patient quality of life by providing faster, more accurate diagnosis and better disease management.

scholarly journals Recombinant Dense Granular Protein (GRA5) for Detection of Human Toxoplasmosis by Western Blot

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Xiao Teng Ching ◽  
Yee Ling Lau ◽  
Mun Yik Fong ◽  
Veeranoot Nissapatorn ◽  
Hemah Andiappan
Keyword(s):  
Western Blot ◽  
Affinity Purification ◽  
Expression System ◽  
Economic Loss ◽  
Serum Samples ◽  
Serological Tests ◽  
Human Patient ◽  
Prokaryotic Expression System ◽  
Human Sera ◽  
Lysate Antigens

Toxoplasma gondiiinfects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of wholeToxoplasmalysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressedT. gondiidense granular protein-5 (GRA5) inEscherichia coliand isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronicT. gondiiinfections (sensitivities of 46.8% and 61.2%, resp.).

scholarly journals Antibodies against Early Proteins of Human Papillomaviruses as Diagnostic Markers for Invasive Cervical Cancer

1998 ◽  
Vol 36 (2) ◽  
pp. 475-480 ◽  
Author(s):  
Wolfgang Meschede ◽  
Klaus Zumbach ◽  
Joris Braspenning ◽  
Martin Scheffner ◽  
Luis Benitez-Bribiesca ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Invasive Cervical Cancer ◽  
Human Papillomaviruses ◽  
Antibody Detection ◽  
Diagnostic Tools ◽  
Immunological Monitoring ◽  
Hpv Dna ◽  
Native Proteins ◽  
Human Sera ◽  
E6 And E7

Cervical cancer is the most prevalent tumor in developing countries and the second most frequent cancer among females worldwide. Specific human papillomaviruses (HPVs) and, most notably, HPV types 16 and 18 are recognized as being causally associated with this malignancy. Antibodies against early HPV proteins E6 and E7 have been found more often in patients with tumors than in controls. Existing peptide enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-E6 and anti-E7 antibodies in human sera have low levels of sensitivity and specificity and thus are not suitable for use as diagnostic tools. Based on highly purified recombinant native proteins, we developed four sandwich ELISAs for the detection of antibodies against HPV type 16 and 18 E6 and E7 proteins. We demonstrate their sensitivities and high degrees of specificity for cervical cancer. Among a total of 501 serum specimens from unselected patients with invasive cervical cancer, 52.9% reacted positively in at least one of the four assays. In contrast, among 244 serum specimens from control subjects without cervical cancer, only 2 reactive serum specimens (0.8%) were found. For 19 of 19 antibody-positive patients, the HPV type indicated by seroreactivity was identical to the HPV DNA type found in the tumor, which also indicates a high degree of specificity for antibody detection with respect to HPV type. In a direct comparison of 72 serum specimens from patients with cervical cancer, 56% of the specimens reacted in at least one of the four protein ELISAs, whereas 40% reacted in at least one of seven peptide ELISAs covering the four antigens. These assays could be of value for the detection of invasive cervical cancer in settings in which cytology-based early tumor screening is not available, for the clinical management of patients diagnosed with cervical cancer, and for the immunological monitoring of E6 and E7 vaccination trials.

scholarly journals Peptide arrays incubated with three collections of human sera from patients infected with mosquito-borne viruses

F1000Research ◽  
2020 ◽  
Vol 8 ◽  
pp. 1875
Author(s):  
Maria del Pilar Martinez Viedma ◽  
Nurgun Kose ◽  
Leda Parham ◽  
Angel Balmaseda ◽  
Guillermina Kuan ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Cell Epitope ◽  
Cross Reactivity ◽  
Human Pathogens ◽  
Peptide Arrays ◽  
Serological Tests ◽  
Array Data ◽  
Prevalence Studies ◽  
Diagnostic Potential ◽  
Human Sera

Background: Global outbreaks caused by emerging or re-emerging arthropod-borne viruses (arboviruses) are becoming increasingly more common. These pathogens include the mosquito-borne viruses belonging to the Flavivirus and Alphavirus genera. These viruses often cause non-specific or asymptomatic infection, which can confound viral prevalence studies. In addition, many acute phase diagnostic tests rely on the detection of viral components such as RNA or antigen. Standard serological tests are often not reliable for diagnosis after seroconversion and convalescence due to cross-reactivity among flaviviruses. Methods: In order to contribute to development efforts for mosquito-borne serodiagnostics, we incubated 137 human sera on individual custom peptide arrays that consisted of over 866 unique peptides in quadruplicate. Our bioinformatics workflow to analyze these data incorporated machine learning, statistics, and B-cell epitope prediction. Results: Here we report the results of our peptide array data analysis, which revealed sets of peptides that have diagnostic potential for detecting past exposure to a subset of the tested human pathogens including Zika virus. These peptides were then confirmed using the well-established ELISA method. Conclusions: These array data, and the resulting peptides can be useful in diverse efforts including the development of new pan-flavivirus antibodies, more accurate epitope mapping, and vaccine development against these viral pathogens.

scholarly journals DEVELOPMENT OF AN IMPROVED EPSTEIN-BARR VIRUS (EBV) NEUTRALIZING ANTIBODY ASSAY TO FACILITATE DEVELOPMENT OF A PROPHYLACTIC GP350-SUBUNIT EBV VACCINE

2020 ◽  
Vol 12 (1) ◽  
pp. e2020016
Author(s):  
Hui Liu ◽  
Lorraine Gemmell ◽  
Rui Lin ◽  
Fengrong Zuo ◽  
Henry H. Balfour ◽  
...  
Keyword(s):  
High Throughput ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Epidemiologic Studies ◽  
Antibody Detection ◽  
Healthy Human ◽  
Barr Virus ◽  
Human Sera ◽  
Epstein Barr ◽  
Bioanalytical Assays

No licensed vaccine is available for prevention of EBV-associated diseases, and robust, sensitive, and high-throughput bioanalytical assays are needed to evaluate immunogenicity of gp350 subunit-based candidate EBV vaccines. Here we have developed and improved analytical tools for such a vaccine’s pre-clinical and clinical validation including a gp350-specific antibody detection assay and an EBV-GFP based neutralization assay for measuring EBV specific antibodies in human donors. The sensitivity of our previously published high-throughput EBV-GFP fluorescent focus (FFA)-based neutralization assay was further improved when guinea pig complement was supplemented using a panel of healthy human sera. Anti-gp350 antibody titers, which were evaluated using an anti-gp350 IgG ELISA assay optimized for capture and detection conditions, were moderately correlated to the FFA-based neutralization titers. Overall, these sensitive, and high-throughput bioanalytical assays are capable of characterizing the serologic response to natural EBV infection, with applications in evaluating EBV antibody status in epidemiologic studies and immunogenicity of candidate gp350-subunit EBV vaccines in clinical studies.

scholarly journals Mechanism of injury of virus-infected cells by antiviral antibody and complement: participation of IgG, F(ab')2, and the alternative complement pathway.

1976 ◽  
Vol 143 (5) ◽  
pp. 1027-1041 ◽  
Author(s):  
L H Perrin ◽  
B S Joseph ◽  
N R Cooper ◽  
M B Oldstone
Keyword(s):  
Measles Virus ◽  
Influenza A ◽  
Igg Antibodies ◽  
Factor B ◽  
Alternative Complement ◽  
Human Sera ◽  
Antiviral Antibody ◽  
Infected Cells ◽  
Virus Influenza

Antibody-mediated C-dependent lysis of cell lines infected with herpes simplex type 1 virus, influenza A degrees virus, measles virus, and mumps virus occurred by the alternative C pathway with the participation of IgG antibodies. Lysis occurred only with immune human sera, Mg++ EGTA immune sera, and immune sera depleted of C4 or treated with Fab anti-C4. Lysis did not occur with nonimmune sera, Mg++ EDTA immune sera, and immune sera heated 50 degrees C for 25 min, depleted of factor B or treated with Fab antifactor B. Lysis was restored to heated and factor B immunodepleted immune sera by addition of factor B, but not by addition of an excess of C2. Further studies showed that lysis of HeLa cells infected with measles virus was induced by both immune IgG and F(ab')2 but not Fab' in the presence of a nonantibody-containing human C source. Lysis of measles virus-infected cells was also indpendent of movement of viral antigens on the surface of the infected cells, as inhibition of viral antigen capping by cytochalasin B or sodium azide was not associated with abrogation of immune lysis.

scholarly journals Standardization of Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assays for Routine Diagnosis of Arboviral Infections

2000 ◽  
Vol 38 (5) ◽  
pp. 1823-1826 ◽  
Author(s):  
Denise A. Martin ◽  
David A. Muth ◽  
Teresa Brown ◽  
Alison J. Johnson ◽  
Nick Karabatsos ◽  
...  
Keyword(s):  
Immunoglobulin M ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Virus Infections ◽  
Virus Family ◽  
Serum Dilution ◽  
Routine Diagnosis ◽  
Human Sera ◽  
Antiviral Antibody ◽  
Antibody Capture

Immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a rapid and versatile diagnostic method that readily permits the combination of multiple assays. Test consolidation is especially important for arthropod-borne viruses (arboviruses) which belong to at least three virus families: the Togaviridae,Flaviviridae, and Bunyaviridae. Using prototype viruses from each of these families and a panel of well-characterized human sera, we have evaluated and standardized a combined MAC-ELISA capable of identifying virus infections caused by members of each virus family. Furthermore, by grouping antigens geographically and utilizing known serological cross-reactivities, we have reduced the number of antigens necessary for testing, while maintaining adequate detection sensitivity. We have determined that a 1:400 serum dilution is most appropriate for screening antiviral antibody, using a positive-to-negative ratio of ≥2.0 as a positive cutoff value. With a blind-coded human serum panel, this combined MAC-ELISA was shown to have test sensitivity and specificity that correlated well with those of other serological techniques.

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