The challenge of identification of autoantibodies specific to systemic autoimmune rheumatic diseases in high throughput operation: proposal of reliable and feasible strategies.

BACKGROUND: Autoantibodies to extractable nuclear antigens (ENA) are good biomarkers for systemic autoimmune rheumatic diseases (SARD), but no one assay for the detection of these antibodies provides satisfactory sensitivity and positive predictive value (PPV). Here we evaluate current assays and propose novel strategies to detect anti-ENA antibodies. METHODS: Diagnostic performance of double immunodiffusion (DID) and several enzyme immunoassays (EIA) for the detection of anti-ENA autoantibodies was determined using samples from 144 patients with a previous clinical diagnosis of SARD and 121 non-autoimmune individuals. A 2-step assay combining EIA and DID was developed and tested on 16,458 serum samples. RESULTS: EIA was more sensitive than DID for all anti-ENA antibodies, but yielded lower PPV (mean=66%) than DID (mean=96%) and a higher percentage of unexpected positive results. ROC-curve guided cut-off adjustments improved PPV for most EIA kits. Using the 2-step assay, over 80% of the samples were screened out by the first step (EIA), with results available within 24h, leaving only about 20% to be confirmed by DID. 2.9% of the 16,485 samples were found to be positive. CONCLUSIONS: A 2-step assay combining the speed and potential for automation of EIA with the high specificity and PPV of DID allows efficient and reliable detection of anti-ENA antibodies. Alternatively, improved PPV can be achieved by adjusting cut-off values for EIA assay results.

Assessing the performance of 3D pharmacophore models in virtual screening: how good are they?

Pharmacophore approaches have evolved to be one of the most successful tools in drug discovery, especially since the past two decades. 3D pharmacophore methods are now commonly used as part of more complex workflows in drug discovery campaigns, and have been successfully and extensively applied in virtual screening (VS) approaches. This review provides a perspective of how to assess the performance of 3D pharmacophore models to be used in VS. Since 3D VS protocols are in general assessed by their ability to discriminate between active and inactive compounds, we summarize the impact of the composition and preparation of modeling and external sets on the outcome of evaluations. Moreover, we highlight the significance of both classic enrichment parameters and advanced descriptors for the performance of 3D pharmacophore-based virtual screening methods.

High-throughput analysis of synthetic peptides for the immunodiagnosis of canine visceral leishmaniasis.

BACKGROUND: Visceral leishmaniasis is the most severe form of leishmaniasis. Approximately 20% of zoonotic human visceral leishmaniasis worldwide is caused by Leishmania infantum, which is also known as Leishmania chagasi in Latin America, and disease incidence is increasing in urban and peri-urban areas of the tropics. In this form of disease, dogs are the main reservoirs. Diagnostic methods used to identify Leishmania infected animals are not able to detect all of the infected ones, which can compromise the effectiveness of disease control. Therefore, to contribute to the improvement of diagnostic methods for canine visceral leishmaniasis (CVL), we aimed to identify and test novel antigens using high-throughput analysis. METHODOLOGY/PRINCIPAL FINDINGS: Immunodominant proteins from L. infantum were mapped in silico to predict B cell epitopes, and the 360 predicted peptides were synthesized on cellulose membranes. Immunoassays were used to select the most reactive peptides, which were then investigated with canine sera. Next, the 10 most reactive peptides were synthesized using solid phase peptide synthesis protocol and tested using ELISA. The sensitivity and specificity of these peptides were also compared to the EIE-LVC Bio-Manguinhos kit, which is recommended by the Brazilian Ministry of Health for use in leishmaniasis control programs. The sensitivity and specificity of the selected synthesized peptides was as high as 88.70% and 95.00%, respectively, whereas the EIE-LVC kit had a sensitivity of 13.08% and 100.00% of specificity. Although the tests based on synthetic peptides were able to diagnose up to 94.80% of asymptomatic dogs with leishmaniasis, the EIE-LVC kit failed to detect the disease in any of the infected asymptomatic dogs. CONCLUSIONS/SIGNIFICANCE: Our study shows that ELISA using synthetic peptides is a technique with great potential for diagnosing CVL; furthermore, the use of these peptides in other diagnostic methodologies, such as immunochromatographic tests, could be beneficial to CVL control programs.