Abstract
The detection of pathogen-specific antibodies remains a cornerstone of clinical diagnostics. Yet, many test exhibit undesirable performance or are completely lacking. Given this, we developed serum epitope repertoire analysis (SERA), a method to rapidly discover conserved, pathogen-specific antigens and their epitopes, and applied it to develop an assay for Chagas disease caused by the protozoan parasite Trypanosoma cruzi. Antibody binding peptide motifs were identified from 28 Chagas repertoires using a bacterial display random 12-mer peptide library and next-generation sequencing (NGS). Thirty-three motifs were selected and mapped to candidate Chagas antigens. In a blinded validation set (n = 72), 30/30 Chagas were positive, 30/30 non-Chagas were negative, and 1/12 Leishmania sp. was positive. After unblinding, a Leishmania cross-reactive epitope was identified and removed from the panel. The Chagas assay exhibited 100% sensitivity (30/30) and specificity (90/90) in a second blinded validation set including individuals with other parasitic infections. Amongst additional epitope repertoires with unknown Chagas serostatus, assay specificity was 99.8% (998/1000). Thus, the Chagas assay achieved a combined sensitivity and specificity equivalent or superior to diagnostic algorithms that rely on three separate tests to achieve high specificity. NGS-based serology via SERA provides an effective approach to discover antigenic epitopes and develop high performance multiplex serological assays.
Highlights
The detection of pathogen-specific antibodies remains a cornerstone of clinical diagnostics
To demonstrate the utility of serum epitope repertoire analysis (SERA) in antigen discovery and multiplex serology we applied SERA to discover conserved immunogenic epitopes of IgG antibodies present in sera from individuals with Chagas disease
The SERA workflow consisted of the steps of (i) separation of antibody-binding peptide library members, (ii) preparation and next-generation sequencing (NGS) of amplicon libraries, (iii) computational discovery of disease-specific motifs and motif panel assembly[15], and (iv) experimental validation of panel performance (Fig. 1)
Summary
The detection of pathogen-specific antibodies remains a cornerstone of clinical diagnostics. Antibody serologic methods typically provide a narrow view of immunity towards a single, or small number of antigens or organisms, while an estimated 1400 pathogens may cause human disease[4]. Many of these diseases lack effective serology tests altogether. We applied serum epitope repertoire analysis (SERA) to discover shared, but highly specific immunogenic epitope motifs associated with Chagas disease caused by the protozoan parasite Trypanosoma cruzi, and thereby develop a serological assay. Our results indicate that NGS-based serology using SERA provides an effective approach to antigen and epitope discovery, and an assay format capable of achieving exceptional diagnostic specificity without multiplexing limitations
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
