High throughput discovery of new serologic biomarkers for Chagas Disease using peptide chips

CARMONA SJ; SCHÄFER-NIELSEN, C; JUAN MUCCI,; ALTCHEH J; TEKIEL V; CAMPETELLA O; A BUSCAGLIA, CARLOS; NIELSEN M; AGÜERO F

Rosario

Congreso; XXVI Reunión Científica Anual; 2013

Sociedad Argentina de Protozoología y Enfermedades Parasitarias

Background. The full set of specificities in a human antibody response to a natural infection remains largely unexplored. Here, we used next-generation high-density peptide microarrays to simultaneously identify and map B-cell epitopes associated to Chagas Disease. Description. The chip consists of a tiling array of ~200K 15-mer peptides synthesized using a maskless photolithographic technique, which in concert cover >500 individual T. cruzi proteins. This represents a coverage of ~5% of the proteome, that includes known antigens, previously uncharacterized proteins selected using a recently published bioinformatic method (Carmona SJ, et al 2012), randomly selected proteins, and random neo-sequences generated based on the T. cruzi di-peptide composition. Antibody pools from healthy and infected individuals were assayed in a single chip, and data were processed to obtain disease-specific signal for each peptide. These were used to reconstruct full-length Ab-binding profiles. A smoothing procedure showed significant improvement on signal to noise ratio. A testing set of Chagas antigens with fine mapped epitopes was used to assess the performance of linear B-cell epitope identification. Performance on this task was excellent, with an area under the ROC curve of 0.92. Discrimination of antigens from non-antigens was done using a threshold of 20u (1u = background interquartile range) and a setup with an antigen/non-antigen ratio of 1%. Using these parameters we were able to detect 20 out of 45 known antigens (44.4%) with a Positive Predictive Value (PPV) of 91% corresponding to 2 false positive predictions. Applying this threshold to all proteins on the chip, we detected 78 new antigens, with an average of 1.5 epitopes/protein.Conclusions. We show that high-density peptide chips allow rapid, high-throughput identification of B-cell epitopes associated to a natural infection. These findings support the use of this technology for high-throughput screenings of serological markers.