Stability Assessment of Four Chimeric Proteins for Human Chagas Disease Immunodiagnosis.

The performance of an immunoassay relies on antigen-antibody interaction; hence, antigen chemical stability and structural integrity are paramount for an efficient assay. We conducted a functional, thermostability and long-term stability analysis of different chimeric antigens (IBMP), in order to assess effects of adverse conditions on four antigens employed in ELISA to diagnose Chagas disease. ELISA-based immunoassays have served as a model for biosensors development, as both assess molecular interactions. To evaluate thermostability, samples were heated and cooled to verify heat-induced denaturation reversibility. In relation to storage stability, the antigens were analyzed at 25 °C at different moments. Long-term stability tests were performed using eight sets of microplates sensitized. Antigens were structurally analyzed through circular dichroism (CD), dynamic light scattering, SDS-PAGE, and functionally evaluated by ELISA. Data suggest that IBMP antigens are stable, over adverse conditions and for over a year. Daily analysis revealed minor changes in the molecular structure. Functionally, IBMP-8.2 and IBMP-8.3 antigens showed reactivity towards anti-T. cruzi antibodies, even after 72 h at 25 °C. Long-term stability tests showed that all antigens were comparable to the control group and all antigens demonstrated stability for one year. Data suggest that the antigens maintained their function and structural characteristics even in adverse conditions, making them a sturdy and reliable candidate to be employed in future in vitro diagnostic tests applicable to different models of POC devices, such as modern biosensors in development.

Novel Genetic Constructs for Production of Recombinant HTLV-1/2 Antigens and Evaluation of Their Reactivity to Plasma Samples from HTLV-1-Infected Patients.

Human T-cell leukemia virus type 1 (HTLV-1) can cause life-threatening diseases for which there are no effective treatments. Prevention of HTLV-1 infection requires massive testing of pregnant women, blood for transfusion, and organs for transplantation, as well as safe sex. In this context, serological assays are widely used for monitoring HTLV-1 infections. Despite the necessity for recombinant antigens to compose serological tests, there is little information available on procedures to produce recombinant HTLV-1/2 antigens for serological diagnostic purposes. In this work, we tested a series of genetic constructions to select those more amenable for production in bacterial systems. To overcome the constraints in expressing sections of viral envelope proteins in bacteria, we have used the p24 segment of the gag protein as a scaffold to display the immunogenic regions of gp46 and gp21. Nine recombinant antigenic proteins derived from HTLV-1 and five derived from HTLV-2 were successfully purified. The HTLV-1 antigens showed high efficiency in discriminating HTLV-positive samples from HTLV-negative samples using enzyme-linked immunosorbent assays. Interestingly, HTLV-1-positive samples showed a high level of cross-reaction with HTLV-2 antigens. This finding is explained by the high sequence conservation between the structural proteins of these two highly related viruses. In summary, the results presented in this work provide a detailed description of the methods used to produce recombinant HTLV-1 and HTLV-2 antigens, and they demonstrate that the HTLV-1 antigens show strong potential for serological diagnosis of HTLV-1 infections.

Correction: Performance of Treponema pallidum recombinant proteins in the serological diagnosis of syphilis.

[This corrects the article DOI: 10.1371/journal.pone.0234043.].

Performance of Treponema pallidum recombinant proteins in the serological diagnosis of syphilis.

Syphilis serodiagnosis is challenging because distinct clinical forms of the infection may influence serological performance and discordant results between tests make clinical decisions difficult. Several recombinant Treponema pallidum-proteins have already been tested for syphilis diagnosis and they are critical to achieve high accuracy in serological testing. Our aim was to assess the varied from performance of T. pallidum-recombinant proteins TmpA, TpN17 and TpN47 for syphilis serodiagnosis. The proteins were evaluated using sera of 338 T. pallidum-negative, 173 T. pallidum-positive individuals and 209 sera from individuals infected with unrelated diseases. The diagnostic potential was validated by analysis of ROC curves. In the liquid microarray analyses, the ROC curve varied from 99.0% for TmpA and TpN17 to 100% for TpN47. The sensitivity score yielded values of up to 90% for TpN17, 100% for TpN47 and 80.0% for TmpA. The lowest and highest specificity values were presented by TpN47 (91.9%) and TmpA antigens (100%), respectively. TpN47 showed the highest accuracy score (95.5%) among all the recombinant proteins assayed. For the ELISA, the ROC curve was 97.2%, 91.8% and 81.6% for TpN17, TmpA and TpN47, respectively. TpN17 and TmpA yielded a sensitivity of 69.9%, while TpN47 obtained a value of 53.8%. Specificity was almost 100% for all three proteins. No cross-reaction was observed for TpN17 in the serum samples from non-bacterial infections. Regarding leptospirosis-positive samples, cross-reactivity score was varied from 8.6 to 34.6%. This is most probably due to conservation of the epitopes in these proteins across bacteria. The use of recombinant proteins in immunoassays for syphilis diagnosis was showed provide greater reliability to results of the treponemal assays. Despite the low sensitivity, the proteins showed high diagnostic capacity due to the AUC values found. However, an improvement in sensitivity could be achieved when antigenic mixtures are evaluated.