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Serological assays are important tools to identify previous exposure to SARS-CoV-2, helping to track COVID-19 cases and determine the level of humoral response to SARS-CoV-2 infections and/or immunization to future vaccines. Here, the SARS-CoV-2 nucleocapsid protein was expressed in Escherichia coli and purified to homogeneity and high yield using a single chromatography step. The purified SARS-CoV-2 nucleocapsid protein was used to develop an indirect enzyme-linked immunosorbent assay for the identification of human SARS-CoV-2 seroconverts. The assay sensitivity and specificity were determined analyzing sera from 140 RT-qPCR-confirmed COVID-19 cases and 210 pre-pandemic controls. The assay operated with 90% sensitivity and 98% specificity; identical accuracies were obtained in head-to-head comparison with a commercial ELISA kit. Antigen-coated plates were stable for up to 3 months at 4 °C. The ELISA method described is ready for mass production and will be an additional tool to track COVID-19 cases.

Chagas disease (CD) serological screening at blood banks is usually performed by a single highly sensitive serological assay, with chemiluminescent immunoassays (CLIAs) being the method of choice. CLIAs employ recombinant, fusion peptides and/or chimeric antigens that selectively capture anti-Trypanosoma cruzi antibodies. However, despite high sensitivity, the ability of these tests to identify CD-positive cases should be evaluated against T. cruzi strains circulating in specific locales. Herein, we used a latent class analysis (LCA) approach employing an array of four chimeric antigens to assess the diagnostic performance of the Liaison XL Murex Chagas CLIA for the detection of anti-T. cruzi IgG in serum samples. The study included a panel of 5014 serum samples collected from volunteer blood donors at the Hematology and Hemotherapy Foundation of the State of Bahia, submitted to anti-T. cruzi antibody detection using Liaison Chagas CLIA and LCA as a reference test in the absence of a gold standard. LCA classified 4993 samples as negative, while positivity for T. cruzi antibodies was predicted in 21 samples. Compared with LCA, CLIA demonstrated sensitivity and specificity of 76.2% and 99.5%, respectively, providing an overall accuracy of 99.4%. In blood banks lacking a de facto highly sensitive screening immunoassay, the low sensitivity offered by Liaison Chagas CLIA renders it unsuitable for standalone use in serological screening procedures for CD. Moreover, blood banks are encouraged to carefully assess the ability of diagnostic methods to identify local T. cruzi strains in circulation.

Despite several available methodologies for Chagas disease (CD) serological screening, the main limitation of chronic CD diagnosis is the lack of effective tools for large-scale screening and point-of-care diagnosis to be used in different CD epidemiological scenarios. Taking into account that developing such a diagnostic tool will significantly improve the ability to identify CD carriers, we aimed at performing a proof-of-concept study (phase I study) to assess the use of these proteins in a point-of-care platform using serum samples from different geographical settings of Brazil and distinct clinical presentations. The diagnostic accuracy study was conducted on a panel of two WHO International Standards (IS) and 14 sera from T. cruzi-positive and 16 from T. cruzi-negative individuals. The results obtained with the test strips were converted to digital images, allowing quantitative comparison expressed as a relative band intensity ratio (RBI). The diagnostic potential and performance were also determined. Regardless of the geographical origin or clinical presentation, all sera with T. cruzi antibodies returned positive both for IBMP-8.1 and IBMP-8.4 chimeric antigens. The area under the ROC curve (AUC) values was 100% for both antigens, demonstrating an outstanding overall diagnostic accuracy (100%). Based on the data, we believe that the lateral flow assays based on these antigens are promising methodologies for screening CD.

Chagas disease (CD) is among the top 10 causes of inability to blood donation. Blood donation centers screen for anti

Background Enzyme-linked immunosorbent assays (ELISA) are generally the chosen test for Chagas disease (CD) diagnosis; however, its performance depends on the antigen preparation adsorbed to the solid phase, which may lead to false-positive results and cross-reactions. The use of chimeric recombinant antigens can overcome this limitation. Four chimeric antigens from Trypanosoma cruzi (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) were developed and evaluated in phase I, II and III studies using indirect ELISA as diagnostic platform. However, peroxidase-labeled secondary anti-human IgG antibody, which is employed in indirect ELISAs, limits its use for the detection of species-specific and class-specific antibodies. To overcome this limitation, peroxidase-labeled antigens can be utilized, diagnosing both acute or chronic infection, in a species and immunoglobulin class-independent manner, through the use of a double-antigen sandwich ELISA (DAgS-ELISA). We aimed to evaluate and validate the diagnostic performance of the chimeric antigens IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 in the DAgS-ELISA platform. Methodology/Principal findings DAgS-ELISA was optimized by checkerboard titration. In phase I study, 207 positive and 205 negative samples were evaluated. Cross-reactivity to other infections was also assessed using 68 samples. The selected conditions for the tests utilized 25 ng of antigen per well and the conjugate diluted at 1:2,000 for all molecules. In the phase I study, the areas under the curve of IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 were 98.7%, 99.5%, 98.6% and 98.8%, respectively. Among the positive samples, IBMP-8.1 antigen classified 53 (25.6%) as false negative, IBMP-8.2, 27 (13%), IBMP-8.3, 24 (11.6%) and IBMP-8.4, 43 (20.8%), giving sensitivities of 74.4%, 87%, 88.4% and 79.2%, respectively. The only antigen that did not reach 100% specificity was IBMP-8.3, with 96.6%. IBMP-8.3 was also the only molecule to show cross-reactivity with HTLV. Conclusions/Significance DAgS-ELISA is a promising tool for immunodiagnosis, and despite the high AUC values, the performance of this assay was different from the values obtained by our group when using these antigens in the indirect ELISA, for this reason, improvements are being considered to increase the sensitivity of the DAgS-ELISA.

Serological assays are important tools to identify previous exposure to SARS-CoV-2, helping to track COVID-19 cases and determine the level of humoral response to SARS-CoV-2 infections and/or immunization to future vaccines. Here the SARS-CoV-2 nucleocapsid protein was expressed in Escherichia coli and purified to homogeneity and high yield using a single chromatography step. The purified SARS-CoV-2 nucleocapsid protein was used to develop an indirect enzyme-linked immunosorbent assay for the identification of human SARS-CoV-2 seroconverts. The assay sensitivity and specificity were determined analyzing sera from 140 PCR-confirmed COVID-19 cases and 210 pre-pandemic controls. The assay operated with 90% sensitivity and 98% specificity; identical accuracies were obtained in head-to-head comparison with a commercial ELISA kit. Antigen coated plates were stable for up to 3 months at 4°C). The ELISA method described is ready to mass production and will be an additional toll to track COVID-19 cases.

The infection caused by the protozoan Trypanosoma cruzi affects humans and is called as Chagas disease. Currently, the main measures available to reduce the incidence of this disease are drug treatment and vector control. Traditionally, the development of vaccines occurs mainly through the use of antigenic candidates of the etiologic agent in the form of a vaccine preparation. Virus-like particles (VLPs) are structures analogous to viral capsids composed essentially of structural proteins and are widely used in vaccination protocols because of their immunostimulatory properties. In this context, the objective of this study was to use strategies in a murine immunization model to characterize the immunostimulatory capacity of VLPs from Triatoma virus (TrV-VLPs), analysed in the presence or absence of the aluminium vaccine adjuvant. In parallel, to characterize the immunogenic behaviour of four T. cruzi chimeric recombinant proteins (mix-IBMP) associated with TrV-VLPs or aluminium vaccine adjuvant., This research was funded by Consejo Superior de Investigaciones Científicas (grant number COOPB20503 - CSIC, Spain) and Global Health and Tropical Medicine [grant number GHTM-UID/multi/04413/2013] and partially supported by grants to DMAG, from the Ministerio de Ciencia e Innovación [BFU2012-36241], Grupos Investigación UPV/EHU 2018 [GIU18/172], and Gobierno Vasco [Elkartek KK-2017/00008], Programa de empresas Innovadoras A1 2020 EKINZAILE, The Basque Country, Spain. This study also was supported by Coordenação de Aperfeiçaomento de Pessoal de Nível Superior (CAPES, Brazil).

In this work, a dual detection system based on an impedimetric immunosensor was developed for the first time for the simultaneous detection of anti-Trypanosoma cruzi and anti-Leishmania infantum antibodies in human and dog serum samples. The IBMP 8.1 and rLci1A/rLci2B recombinant antigens were immobilized over the surface of dual screen-printed carbon electrodes (W1 and W2) modified with poly (4-hydroxyphenylacetic acid). Under optimized conditions, the immunosensor recognized specific interactions for anti-T. cruzi antibodies up to a dilution of 1:10,240 and for anti-L. infantum up to 1:5120 in canine serum samples. Relative standard deviation (RSD) values of 2.8% for W1 and 3.6% for W2 were obtained for T. cruzi (W1) and L. infantum antigen (W2) samples in three different electrodes for 3 days (n = 9). The immunosensor was stored at 4 °C for 8 weeks, with activity retention of 70.2% in W1 and 78.2% in W2. The results using the recombinant proteins revealed that all antigens discriminated between negative and positive samples (p

The existence of an imperfect reference standard presents complications when evaluating the unbiased performance of novel diagnostic techniques. This is especially true in the absence of a gold standard, as is the case in chronic Chagas disease (CD) diagnosis. To circumvent this constraint, we elected to use latent class analysis (LCA). Previously, our group demonstrated the high performance of four Trypanosoma cruzi–chimeric proteins (Molecular Biology Institute of Paraná [IBMP]-8.1, -8.2, -8.3, and -8.4) for CD diagnosis using several distinct immunoassays. Although commercial tests had previously been established as a reference standard, the diagnostic performance of these chimeric antigens could present bias because these tests fail to produce 100% accurate results. Thus, we used LCA to assess the performance of these IBMP chimeric antigens in chronic CD diagnosis. Using the LCA model as a gold standard, sensitivity and specificity values ranged from 93.5% to 99.4% and 99.6% to 100%, respectively. The accuracy values were 96.2% for IBMP-8.2, approximately 98% for IBMP-8.1 and IBMP-8.3, and nearly 100% for IBMP-8.4. For IBMP-8.1 and IBMP-8.2, higher positive predictive values were associated with increases in hypothetical prevalence. Similarly, higher hypothetical prevalence resulted in lower negative predictive values for IBMP-8.1, IBMP-8.2, and IBMP-8.3. In addition, samples with serodiscordant results from commercial serological tests were analyzed using LCA. Molecular Biology Institute of Paraná -8.1 demonstrated potential for use in confirmatory testing with regard to samples with inconsistent results. Moreover, our findings further confirmed the remarkable performance of the IBMP-8.4 antigen to diagnose chronic CD in both endemic and non-endemic areas.

Chimeric T. cruzi antigens have been proposed as a diagnostic tool for chronic Chagas disease (CD) in both settings where Chagas disease is endemic and those where it is not endemic. Antibody response varies in accordance to each T. cruzi strain, presenting challenges to the use of antigens lacking demonstrated cross-reactivity with Leishmania spp., Chimeric T. cruzi antigens have been proposed as a diagnostic tool for chronic Chagas disease (CD) in both settings where Chagas disease is endemic and those where it is not endemic. Antibody response varies in accordance to each T. cruzi strain, presenting challenges to the use of antigens lacking demonstrated cross-reactivity with Leishmania spp. Our group expressed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) and previously assessed their diagnostic performance to determine cross-reactivity with Leishmania spp. Here, we validated our findings using serum samples from different Brazilian geographic areas reporting endemic Chagas disease, endemic visceral or American cutaneous leishmaniasis (ACL), or both. Overall, 829 serum samples were evaluated using commercial and IBMP enzyme-linked immunosorbent assays. Due to the absence of a reference assay to diagnosis CD, latent class analysis (LCA) was performed through the use of a statistical model. The incidence of cross-reactivity for ACL-positive samples varied from 0.35% (IBMP-8.3) to 0.70% (IBMP-8.1 and IBMP-8.2). Regarding visceral leishmaniasis (VL)-positive samples, the IBMP-8.2 and IBMP-8.3 antigens cross-reacted with six (3.49%) and with only one sample (0.58%), respectively. No cross-reactivity with either ACL or VL was observed for the IBMP-8.4 antigen. Similarly, no cross-reactions were found when VL-positive samples were assayed with IBMP-8.1. The agreement among the results obtained using IBMP antigens ranged from 97.3% for IBMP-8.2 and 99% for IBMP-8.1 and IBMP-8.3 to 100% for IBMP-8.4, demonstrating almost perfect agreement with LCA. Accordingly, in light of the negligible cross-reactivity with both ACL and VL, we suggest the use of IBMP antigens in regions where T. cruzi and Leishmania spp. are coendemic.

Background Dogs are considered sentinels in areas of Trypanosoma cruzi transmission risk to humans. ELISA is generally the method of choice for diagnosing T. cruzi exposure in dogs, but its performance substantially depends on the antigenic matrix employed. In previous studies, our group has developed four chimeric antigens (IBMP-8.1, 8.2, 8.3, and 8.4) and evaluated their potential for diagnosing T. cruzi exposure in humans. For human sera, these chimeric antigens presented superior diagnostic performances as compared to commercial tests available in Brazil, Spain, and Argentina. Therefore, in this study we have evaluated the potential of these antigenic proteins for detection of anti-T. cruzi IgG antibodies in dog sera. Methodology/Principal findings The IBMP-ELISA assays were optimized by checkerboard titration. Subsequently, the diagnostic potential was validated through analysis of ROC curves and the performance of the tests was determined using double entry tables. Cross-reactivity was also evaluated for babesiosis, ehrlichiosis, dirofilariosis, anaplasmosis, and visceral leishmaniasis. Best performance was shown by IBMP-8.3 and IBMP-8.4, although all four antigens demonstrated a high diagnostic performance with 46 positive and 149 negative samples tested. IBMP-8.3 demonstrated 100% sensitivity, followed by IBMP-8.4 (96.7–100%), IBMP-8.2 (73.3–87.5%), and IBMP-8.1 (50–100%). The highest specificities were achieved with IBMP-8.2 (100%) and IBMP-8.4 (100%), followed by IBMP-8.3 (96.7–97.5%) and IBMP 8.1 (89.1–100%). Conclusions/Significance The use of chimeric antigenic matrices in immunoassays for anti-T. cruzi IgG antibody detection in sera of infected dogs was shown to be a promising tool for veterinary diagnosis and epidemiological studies. The chimeric antigens used in this work allowed also to overcome the common hurdles related to serodiagnosis of T. cruzi infection, especially regarding variation of efficiency parameters according to different strains and cross-reactivity with other infectious diseases., Author summary Despite dogs being considered T. cruzi’s most important domestic sentinel/reservoir and also suffering from the outcomes of the infection, there has never been a commercially available test to diagnose T. cruzi infection in dogs. As such, our group’s objective was to develop a state-of-the-art serological diagnostic test utilizing four chimeric antigens (IBMP-8.1, 8.2, 8.3, and 8.4) in an ELISA platform to accurately identify canine anti-T. cruzi IgG antibodies. The IBMP-ELISA assays were optimized, evaluated for cross-reactivity towards multiple canine parasite’s, including Leishmania spp., its diagnostic potential was validated and the test’s performance was determined using double entry tables. The IBMP-8.3 antigen demonstrated 100% sensitivity, followed by IBMP-8.4, whereas the highest specificities were achieved with IBMP-8.2 (100%) and IBMP-8.4 (100%). Therefore, we’ve concluded that the serodiagnosis through anti-T. cruzi IgG detection in dogs, utilizing chimeric antigenic matrices in immunoassays is a promising tool for veterinary diagnosis and epidemiological surveillance. Furthermore, the use of chimeric antigens efficiently addressed common hurdles related to T. cruzi serodiagnosis, especially regarding efficiency variation in response to different strains and cross-reactivity.

Background Laboratory diagnosis of chronic Chagas disease is a troubling factor due to lack of reference tests. The WHO suggests the use of two distinct commercial serological tests in parallel. The performance of commercial immunoassays might fluctuate depending on the antigenic matrices and the local strains of T. cruzi in different geographical settings. The use of antigenic matrices based on chimeric proteins can solve these limitations. Here, we evaluated the diagnostic performance of two chimeric T. cruzi antigens (IBMP-8.1 and -8.4) to diagnose chronic Chagas disease in individuals from endemic South American countries. Methodology/Principal findings IBMP-8.1 and IBMP-8.4 chimeric antigens were expressed as soluble proteins in E. coli and purified using chromatography methods. Reactivity of IBMP-8.1 and IBMP-8.4 was assessed using an in-house ELISA with sera from 122 non-infected and 215 T. cruzi-infected individuals from Argentina, Bolivia, and Paraguay. Cut-off values were based on ROC curves and performance parameters were determined using a dichotomous approach. Area under the curve values were > 99.7% for both IBMP-8.1 and IBMP-8.4 antigens. IgG levels in T. cruzi-positive and negative samples were higher for IBMP-8.4 than IBMP-8.1. Both IBMP-8.1 and -8.4 were 100% specific, while IBMP-8.4 were 100% sensitive compared to IBMP-8.1 (95.3%). Admitting RI values of 1.0 ± 0.10 as the inconclusive interval, 6.2% of the samples tested using IBMP-8.1 and 2.1% using IBMP-8.4 fell inside the grey zone. Based on accuracy and diagnostic odds ratio values, IBMP-8.4 presented the best performance. Differences in sensitivity and IgG levels among the samples from Argentina, Bolivia, and Paraguay were not significant. Conclusions/Significance Our findings showed a notable performance of IBMP-8.1 and -8.4 chimeric antigens in diagnosing chronic Chagas disease in individuals from endemic South American countries, confirming our hypothesis that these antigens could be used in geographical areas where distinct T. cruzi DTUs occur. Fil: Pimenta Del Rei, Rodrigo. Faculty of Technology and Sciences of Bahia; Brasil Fil: Leony, Leonardo Maia. Fundación Oswaldo Cruz; Brasil Fil: Fiorani Celedon, Paola Alejandra. Molecular Biology Institute of Parana; Brasil Fil: Zanchin, Nilson Ivo Tonin. Fundación Oswaldo Cruz; Brasil Fil: Galvão dos Reis, Mitermayer. Fundación Oswaldo Cruz; Brasil. Federal University of Bahia; Brasil. University of Yale; Estados Unidos Fil: de Miranda Gomes, Yara. Fundación Oswaldo Cruz; Brasil Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Neves Santos, Fred Luciano. Fundación Oswaldo Cruz; Brasil

Background Canine Visceral leishmaniasis (CVL) is a serious public health problem, thus for its control, the Ministry of Health in Brazil recommends the rapid diagnosis and euthanasia of seropositive dogs in endemic areas. Therefore, our group had previously selected six recombinant proteins (rLci1, rLci2, rLci4, rLci5, rLci8, and rLci12) due to their high potential for CVL diagnostic testing. The present study aims to produce an immunodiagnostic test using the aforementioned antigens, to improve the performance of the diagnosis of CVL recommended by Brazilian Ministry of Health. Methodology/Principal findings To evaluate the recombinant proteins in the serological assays, positive and negative samples were selected based on parasitological test (culture) and molecular test (qPCR) of splenic aspirate. Initially, we selected 135 dog serum samples, 73 positives (symptomatic and asymptomatic) and 62 negatives to screen recombinant proteins on ELISA platform. Then, for rLci5 ELISA validation, 361 serum samples collected in a cross-sectional study were selected, being 183 positives (symptomatic and asymptomatic) and 178 negatives. In the screening of the recombinant proteins, rLci5 was the only protein to present a performance statistically higher than the performance presented by EIE-LVC test, presenting 96% (IC 95%; 85–99%) vs. 83% (IC 95%; 69–92%) of sensitivity for symptomatic dogs, 71% (IC 95%; 49–97%) vs. 54% (IC 95%; 33–74%) for asymptomatic dogs and 94% (IC 95%; 83–99%) vs, 88% (IC 95%; 76–95% of specificity. Thus, the rLci5 protein was selected to compose a final ELISA test. Validation of rLci5 ELISA showed 87% (IC 81–91%) of sensitivity, 94% (IC 95%; 90–97%) of specificity and 90% accuracy. Testing the EIE-LVC with the same validation panel, we observed a lower performance when compared to ELISA rLci5 (sensitivity of 67% (IC 95%; 59–74%), specificity of 87% (IC 95%; 81–92%), and accuracy of 77%). Finally, the performance of current CVL diagnostic protocol recommended by Brazilian Ministry of Health, using DPP-LVC as screening test and EIE-LVC as confirmatory test, was compared with a modified protocol, replacing EIE-LVC by rLci5 ELISA. The current protocol presented a sensitivity of 59% (IC 95%; 52–66%), specificity of 98% (IC 95%; 95–99%) and accuracy of 80% (IC 95%; 76–84%), while the modified protocol presented a sensitivity of 71% (IC 95%; 63–77%), specificity of 99% (IC 95%; 97–100%) and accuracy of 86% (IC 95%; 83–89%). Conclusion Thus, we concluded that rLci5 ELISA is a promising test to replace EIE-LVC test and increase the diagnostic performance of CVL in Brazil., Author summary Visceral leishmaniasis is a tropical disease caused by the protozoan parasite Leishmania infantum, which is transmitted to humans through the bite of infected sand flies. Infected dogs are considered the main urban reservoir of parasite. Identification and euthanasia of infected dogs is one of the main strategies for VL control recommended by the Brazilian Ministry of Health. Hence, to improve the efficacy of control measures, it is essential an accurate diagnose of naturally infected dogs in endemic areas. Herein, we sought to produce a new immunodiagnostic test using recombinant antigens of Leishmania infantum to improve the performance of the diagnosis of CVL recommended by Brazilian Ministry of Health.

Diagnosing chronic Chagas disease (CD) requires antibody–antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi -specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti- T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania , a pathogen with high similarity to T. cruzi , showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland–Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD.

Background The performance of current serologic tests for diagnosing chronic Chagas disease (CD) is highly variable. The search for new diagnostic markers has been a constant challenge for improving accuracy and reducing the number of inconclusive results. Methodology/Principal findings Here, four chimeric proteins (IBMP-8.1 to -8.4) comprising immunodominant regions of different Trypanosoma cruzi antigens were tested by enzyme-linked immunosorbent assay. The proteins were used to detect specific anti-T. cruzi antibodies in the sera of 857 chagasic and 689 non-chagasic individuals to evaluate their accuracy for chronic CD diagnosis. The antigens were recombinantly expressed in Escherichia coli and purified by chromatographic methods. The sensitivity and specificity values ranged from 94.3% to 99.3% and 99.4% to 100%, respectively. The diagnostic odds ratio (DOR) values were 6,462 for IBMP-8.1, 3,807 for IBMP-8.2, 32,095 for IBMP-8.3, and 283,714 for IBMP-8.4. These chimeric antigens presented DORs that were higher than the commercial test Pathozyme Chagas. The antigens IBMP-8.3 and -8.4 also showed DORs higher than the Gold ELISA Chagas test. Mixtures with equimolar concentrations were tested in order to improve the diagnosis accuracy of negative samples with high signal and positive samples with low signal. However, no gain in accuracy was observed relative to the individual antigens. A total of 1,079 additional sera were used to test cross-reactivity to unrelated diseases. The cross-reactivity rates ranged from 0.37% to 0.74% even for Leishmania spp., a pathogen showing relatively high genome sequence identity to T. cruzi. Imprecision analyses showed that IBMP chimeras are very stable and the results are highly reproducible. Conclusions/Significance Our findings indicate that the IBMP-8.4 antigen can be safely used in serological tests for T. cruzi screening in blood banks and for chronic CD laboratory diagnosis., Author summary Chagas disease infection is a debilitating and neglected disease that occurs in 21 Latin America countries. The laboratory diagnosis is based on finding specific antibodies against Trypanosoma cruzi pathogen. Several methods are available currently, but their performance depends on matrix used as antigen. In general, two methodologies are required to perform a correct diagnosis. We tested four chimeric proteins comprised by various antigens of T. cruzi by a method designated enzyme-linked immunosorbent assay. The proteins were high sensitivities and specifics, presenting accuracy higher than the commercial test Pathozyme Chagas. Additionally, the proteins IBMP-8.3 and -8.4 were more accurate than the Gold ELISA Chagas test. The proteins presented a very low cross-reactivity to unrelated diseases, including Leishmania spp. Reproducibility analyses showed that IBMP proteins are stable and the results are highly reproducible. Our findings indicate that the IBMP-8.4 protein can be safely used in serological tests for T. cruzi screening in blood banks and chronic CD laboratory.

The use of conditioned medium (CM) from human cardiac explants (HCEs) as a potential source of paracrine factors for adult stem cell signaling has never been evaluated. We hypothesized that HCEs might provide a source of soluble factors triggering the differentiation of mesenchymal stem cells (MSCs) into cardiomyocyte-like cells. By using two-dimensional electrophoresis (2-DE) gels/mass spectrometry and antibody macroarray assays, we found that HCEs release macromolecules, including cytokines, growth factors and myocardial and metabolism-related proteins into the culture medium. We identified a total of 20 proteins in the HCE-CM. However, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-DE, these 20 proteins account for only a fraction of the total number of proteins present in the HCE-CM. We also found that CM increased the proliferation of bone marrow-derived-MSCs (BM-MSCs) in vitro. Unlike the other effects, this effect was most evident after 48 h of culture. Moreover, we examined the effect of HCE-CM on levels of mRNA and protein for specific cardiac markers. We showed that a surprisingly big fraction of BM-MSCs (3.4–5.0%) treated in vitro with HCE-CM became elongated and began to express cardiac markers, consistent with their possible differentiation into cardiomyocyte-like cells. Our in vitro model may be useful not only per se, but also for studies of the mechanisms of action of soluble factors involved in cell differentiation, paving the way for possible new protein-based treatments in the future.

Recent advances in genomics and proteomics have provided an excellent opportunity to understand complex biological processes such as wood formation at the gene and protein levels. The aim of this work was to describe the proteins participating in the processes involved in juvenile wood formation by isolating proteins from the cambial region of Eucalyptus grandis, at three ages of growth (6-month-old seedlings, 3- and 6-year-old trees), and also to identify proteins differentially expressed. Using a 2-D-LC-MS/MS strategy we identified a total of 240 proteins, with 54 corresponding spots being present in at least two ages. Overall, nine proteins classified into the functional categories of metabolism, cellular processes, and macromolecular metabolism showed significant changes in expression. Proteins were classified into seven main functional categories, with metabolism representing 35.2% of the total proteins identified. The comparison of the reference maps showed not only differences in the expression pattern of individual proteins at each age, but also among isoforms. The results described in this paper provide a dynamic view of the proteins involved in the formation of juvenile wood in E. grandis.

A proteômica expandiu-se dentro da área biológica a partir da década de 90 com o desenvolvimento de técnicas de ionização branda que permitem analisar macromoléculas por espectrometria de massas (MS) e é uma nova estratégia na busca de genes de interesse e de informações sobre o controle da expressão gênica para a manipulação genética de plantas. Na busca por genes diferencialmente expressos durante o desenvolvimento da madeira, foi estabelecida uma plataforma 2D-LC-MS/MS (eletroforese bidimensional seguida de cromatografia líquida e espectrometria de massas em tandem) e identificadas 72 proteínas da região cambial de Eucalyptus grandis de árvores de 2 anos e 8 meses. A identificação foi feita por homologia com o banco de proteínas SwissProt, com um banco de ESTs específico de Eucalyptus spp. (GENOLYPTUS) e com o NCBI. Os dados de proteínas também foram confrontados com a expressão dos transcritos correspondentes obtidos a partir do mesmo tecido por SAGE (Serial Analysis of Gene Expression). Dentre as proteínas identificadas, a maior parcela (24,1 %) tem função relacionada com resposta a estresse, 20,5 % pertence a metabolismo de energia, 17,8 % são componentes estruturais, 14,3 % participam em metabolismo de macromoléculas e 15,2 % em metabolismos básicos como de nitrogênio, aminoácidos, nucleotídeos, lipídeos e metabolismo secundário. Proteomics impacted the biological sciences since the 90´s after the development of soft ionization techniques that allow to analyse macromolecules by mass spectrometry (MS). It became a new strategy to identify genes and obtain information about the molecular control of gene expression, of importance to genetic manipulation of plants. In order to search for genes involved in wood quality and to understand gene expression during wood development, a 2D-LC-MS/MS system (bidimensional electrophoresis followed by liquid cromatography coupled to mass spectrometry in tandem) was stablished. 72 proteins from the cambial region of Eucalyptus grandis trees at the age of 2 years and 8 months were identified. The MS/MS spectra were processed using the SwissProt databank, an EST (Expressed Sequence Tags) bank of Eucalyptus spp. (GENOLYPTUS) and the NCBI. A comparation of gene and protein expression was carried out using a databank constructed in our laboratory using SAGE (Serial Analysis of Gene Expression) obtained from the same cambial tissue. Most identified proteins are related to stress response (24.1 %), 20.5 % participate in energy metabolism, 17.8 % are important to the cell as structural components, 14,3 % are related to macromolecular metabolism and 15,2 % to basic metabolism of nitrogen, amino acids, nucleotides, lipids and secondary metabolism.

The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections. Here, we describe the production of four chimeric T. cruzi proteins and the assessment of their performance for diagnostic purposes. Circular Dichroism spectra indicated the absence of well-defined secondary structures, while polydispersity evaluated by Dynamic Light Scattering revealed only minor aggregates in 50 mM carbonate-bicarbonate (pH 9.6), demonstrating that it is an appropriate buffering system for sensitizing microplates. Serum samples from T. cruzi-infected and non-infected individuals were used to assess the performance of these antigens for detecting antibodies against T. cruzi, using both enzyme-linked immunosorbent assay and a liquid bead array platform. Performance parameters (AUC, sensitivity, specificity, accuracy and J index) showed high diagnostic accuracy for all chimeric proteins for detection of specific anti-T. cruzi antibodies and differentiated seropositive individuals from those who were seronegative. Our data suggest that these four chimeric proteins are eligible for phase II studies.

Background The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches. Results We constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway® technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the c-myc epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (Tc Rab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed. Conclusions We constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.

Despite the importance of Eucalyptus spp. in the pulp and paper industry, functional genomic approaches have only recently been applied to understand wood formation in this genus. We attempted to establish a global view of gene expression in the juvenile cambial region of Eucalyptus grandis Hill ex Maiden. The expression profile was obtained from serial analysis of gene expression (SAGE) library data produced from 3- and 6-year-old trees. Fourteen-base expressed sequence tags (ESTs) were searched against public Eucalyptus ESTs and annotated with GenBank. Altogether 43,304 tags were generated producing 3066 unigenes with three or more copies each, 445 with a putative identity, 215 with unknown function and 2406 without an EST match. The expression profile of the juvenile cambial region revealed the presence of highly frequent transcripts related to general metabolism and energy metabolism, cellular processes, transport, structural components and information pathways. We made a quantitative analysis of a large number of genes involved in the biosynthesis of cellulose, pectin, hemicellulose and lignin. Our findings provide insight into the expression of functionally related genes involved in juvenile wood formation in young fast-growing E. grandis trees.