High-throughput Identification of DNA-Encoded IgG Ligands that Distinguish Active and Latent Mycobacterium tuberculosis Infections
| Autor: | Irena Suponitsky-Kroyter, Katharina Ronacher, Patrick J. McEnaney, Gerhard Walzl, Ofelia Utset, Andrew B. MacConnell, Kim Stanley, Brian M. Paegel, Marie L. Malone, Thomas Kodadek, Kimberly R. Mendes, Todd M. Doran, Valerie Cavett, John Maina Ndungu |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Ligands 01 natural sciences Biochemistry Article Deep sequencing Epitope Mycobacterium tuberculosis Epitopes Structure-Activity Relationship 03 medical and health sciences Bacterial Proteins Antigen Antibody Repertoire Latent Tuberculosis Peptide Library Escherichia coli Humans Peptide library Tuberculosis Pulmonary Pathogen Solid-Phase Synthesis Techniques Antigens Bacterial biology 010405 organic chemistry DNA General Medicine biology.organism_classification Virology High-Throughput Screening Assays 0104 chemical sciences 3. Good health 030104 developmental biology Immunoglobulin G biology.protein Molecular Medicine Antibody Oligopeptides Acyltransferases |
| Zdroj: | ACS Chemical Biology. 12:234-243 |
| ISSN: | 1554-8937 1554-8929 |
| Popis: | The circulating antibody repertoire encodes a patient's health status and pathogen exposure history, but identifying antibodies with diagnostic potential usually requires knowledge of the antigen(s). We previously circumvented this problem by screening libraries of bead-displayed small molecules against case and control serum samples to discover “epitope surrogates” (ligands of IgGs enriched in the case sample). Here, we describe an improved version of this technology that employs DNA-encoded libraries and high-throughput FACS-based screening to discover epitope surrogates that differentiate noninfectious/latent (LTB) patients from infectious/active TB (ATB) patients, which is imperative for proper treatment selection and antibiotic stewardship. Normal control/LTB (10 patients each, NCL) and ATB (10 patients) serum pools were screened against a library (5 × 106 beads, 448k unique compounds) using fluorescent anti-human IgG to label hit compound beads for FACS. Deep sequencing decoded all hit structures and each hit's occurrence frequencies. ATB hits were pruned of NCL hits and prioritized for resynthesis based on occurrence and homology. Several structurally homologous families were identified and 16/21 resynthesized representative hits validated as selective ligands of ATB serum IgGs (p < 0.005). The native secreted TB protein Ag85B (though not the E. coli recombinant form) competed with one of the validated ligands for binding to antibodies, suggesting that it mimics a native Ag85B epitope. The use of DNA-encoded libraries and FACS-based screening in epitope surrogate discovery reveals thousands of potential hit structures. Distilling this list down to several consensus chemical structures yielded a diagnostic panel for ATB composed of thermally stable and economically produced small molecule ligands in place of protein antigens. |
| Databáze: | OpenAIRE |
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