title: Hyperthermostable binding molecules on phage: Assay components for point-of-care diagnostics for active tuberculosis infection.
abstract: Tuberculosis is the leading cause of death from infectious disease worldwide. The low sensitivity, extended processing time, and high expense of current diagnostics are major challenges to the detection and treatment of tuberculosis. Mycobacterium tuberculosis ornithine transcarbamylase (Mtb OTC, Rv1656) has been identified in the urine of patients with active TB infection and is a promising target for point-of-care diagnostics. Specific binding proteins with low nanomolar affinities for Mtb OTC were selected from a phage display library built upon a hyperthermostable Sso7d scaffold. Phage particles displaying Sso7d variants were utilized to generate a sandwich ELISA-based assay for Mtb OTC. The assay response is linear between 2 ng/mL and 125 ng/mL recombinant Mtb OTC and has a limit of detection of 400 pg/mL recombinant Mtb OTC. The assay employing a phage-based detection reagent is comparable to commercially-available antibody-based biosensors. Importantly, the assay maintains functionality at both neutral and basic pH in presence of salt and urea over the range of concentrations typical for human urine. Phage-based diagnostic systems may feature improved physical stability and cost of production relative to traditional antibody-based reagents, without sacrificing specificity and sensitivity.
authors.affiliation: Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, CO 80523, United States.
authors.forename: Ning
authors.initials: N
authors.lastname: Zhao
authors.affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, United States.
authors.forename: John
authors.initials: J
authors.lastname: Spencer
authors.affiliation: Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, CO 80523, United States.
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Schmitt
authors.affiliation: Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, CO 80523, United States; Department of Chemistry, Colorado State University, Fort Collins, CO 80523, United States; School of Biomedical Engineering, Colorado State University, Fort Collins, CO 80523, United States. Electronic address: nickfisk@colostate.edu.
authors.forename: John D
authors.initials: JD
authors.lastname: Fisk
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D019843:Archaeal Proteins / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D061505:Cell Surface Display Techniques / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D019151:Peptide Library; D019095:Point-of-Care Systems; D017386:Sequence Homology, Amino Acid; D048229:Sulfolobus solfataricus / Q000378:metabolism; D013696:Temperature; D014376:Tuberculosis / Q000175:diagnosis* / Q000378:metabolism / Q000382:microbiology
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D019151:Peptide Library; C093228:Sso7d protein, Sulfolobus
keywords: Alternative scaffold; Diagnostics; Molecular recognition; Mycobacterium tuberculosis; Ornithine transcarbamylase; Phage display