We have used high-density peptide arrays displaying short peptides to interrogate the specificity of the antibody repertoires of Chagas Disease patients across the Americas. The detailed description of our work can be seen in our original paper.
Chagas Disease (American trypanosomiasis) is a lifelong infection caused by the protozoan parasite Trypanosoma cruzi. Despite being discovered ~100 years ago, the condition remains a major social and public health problem in the Americas and is regarded as a neglected tropical disease by the World Health Organization.
Trypanosoma cruzi is a unicellular eukaryote that infects and replicates within cells. There are 6-7 evolutionary lineages of the parasite across the Americas, circulating in different ecoepidemiological cycles (domestic, peridomestic, sylvatic). All cause human infections, with a diversity of clinical manifestations.
Peptide arrays display short peptides at addressable positions. A very high-density of probes is achieved by in situ peptide synthesis driven by digital light processors. Such arrays contain hundreds of thousands to millions of peptides.
The term “antibody repertoire” refers to the entire set of antibodies produced by an individual as part of the adaptive immune response to infections or vaccines.
We assayed high density arrays displaying immobilized short peptides (candidate antigens and epitopes) with the same methodology used in indirect immunoassays. Arrays were first incubated with a primary antibody (human serum sample or pool of samples), then washed to remove unbound immunoglobulins. Arrays were next incubated with a secondary antibody, fluorescently labeled. This secondary antibody binds to human immunoglobulin G (total IgG). After washing to remove unbound antibodies, fluorescence was read in a scanner.
The discovery screening was aimed at displaying the complete proteome of T. cruzi using short peptides; and detecting which of these peptides were bound by antibodies by subjects with Chagas disease. Two complete proteomes were displayed at this stage (2.84 million peptides), derived from T. cruzi strains CL-Brener and Sylvio X10, representative of lineages TcI (Sylvio X10) and TcVI (CL-Brener, hybrid). To maximize coverage for discovery, each peptide array was incubated with a pool of 5-6 serum samples.