title: Identification and characterization of a peptide affinity reagent for detection of noroviruses in clinical samples.
abstract: Norovirus (NoV) is the most common agent of nonbacterial epidemic gastroenteritis and is estimated to cause 21 million cases of the disease in the United States annually. The antigen enzyme-linked immunosorbent assays (ELISAs) currently available for NoV diagnosis detect only certain strains and are approved for use in the United States only in epidemics where NoV is suspected. There is a clear need for simpler, more rapid, and more reliable diagnostic tools for the detection of NoV. In this study, phage display technology was used to screen a library of phage displaying random 12-mer peptides for those that bind to Norwalk virus virus-like particles (NV VLPs). Three phage clones displaying unique peptides were identified, and both the peptide-displaying phages and the peptides were confirmed to bind specifically to NV VLPs. The peptide displayed on phage clone NV-N-R5-1 was determined to bind to the protruding domain of the VP1 capsid protein. This phage also bound to NV VLPs seeded into NoV-negative stool with a limit of detection of 1.56 ng NV VLP. This value was comparable to monoclonal antibody (MAb) 3912, which is currently used in commercially available assays. Furthermore, the NV-N-R5-1 phage exhibited high specificity by detecting NV only in previously characterized NV-positive stool samples in contrast to no detection in NV-negative stool samples. These data demonstrate that the further development of NV-N-R5-1 phage as a diagnostic reagent is possible and might offer several distinct advantages over antibodies, such as decreases in the time and cost of production and ease of isolating phage against other epidemic strains currently circulating as well as those that are emerging.
authors.affiliation: Departments of Pharmacology, aylor College of Medicine, Houston, Texas, USA.
authors.forename: Jennifer D
authors.initials: JD
authors.lastname: Rogers
authors.forename: Nadim J
authors.initials: NJ
authors.lastname: Ajami
authors.forename: Bartlomiej G
authors.initials: BG
authors.lastname: Fryszczyn
authors.forename: Mary K
authors.initials: MK
authors.lastname: Estes
authors.forename: Robert L
authors.initials: RL
authors.lastname: Atmar
authors.forename: Timothy
authors.initials: T
authors.lastname: Palzkill
mesh_terms: D017250:Caliciviridae Infections / Q000175:diagnosis* / Q000821:virology; D019411:Clinical Laboratory Techniques / Q000379:methods*; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D029322:Norovirus / Q000302:isolation & purification*; D019151:Peptide Library; D010455:Peptides / Q000302:isolation & purification; D011485:Protein Binding; D012680:Sensitivity and Specificity; D014481:United States; D014773:Virology / Q000379:methods*
chemical_list: D019151:Peptide Library; D010455:Peptides
references.citation: J Virol Methods. 2013 Jan;187(1):121-6
references.pmid: 23022731
references.citation: Emerg Infect Dis. 2008 Oct;14(10):1553-7
references.pmid: 18826818
references.citation: Science. 1999 Oct 8;286(5438):287-90
references.pmid: 10514371
references.citation: Clin Diagn Lab Immunol. 1999 Jan;6(1):142-5
references.pmid: 9874680
references.citation: JAMA. 2012 Oct 24;308(16):1668-75
references.pmid: 23079758
references.citation: J Infect Dis. 1998 Dec;178(6):1571-8
references.pmid: 9815206
references.citation: J Virol Methods. 2012 Jul;183(1):63-8
references.pmid: 22575687
references.citation: Methods Mol Biol. 1996;66:149-69
references.pmid: 8959713
references.citation: J Virol. 1992 Nov;66(11):6527-32
references.pmid: 1328679
references.citation: Appl Microbiol Biotechnol. 2006 Mar;70(1):2-11
references.pmid: 16365766
references.citation: N Engl J Med. 2009 Oct 29;361(18):1776-85
references.pmid: 19864676
references.citation: N Engl J Med. 2012 Nov 29;367(22):2126-32
references.pmid: 23190223
references.citation: J Virol. 2005 Jun;79(12):7402-9
references.pmid: 15919896
references.citation: Bacteriophage. 2012 Apr 1;2(2):105-283
references.pmid: 23050221
references.citation: Emerg Infect Dis. 2004 Oct;10(10):1827-34
references.pmid: 15504271
references.citation: Chembiochem. 2003 Jan 3;4(1):14-25
references.pmid: 12512072
references.citation: J Clin Microbiol. 2002 Nov;40(11):4244-50
references.pmid: 12409405
references.citation: Clin Microbiol Rev. 2001 Jan;14(1):15-37
references.pmid: 11148001
references.citation: J Clin Virol. 2011 Jun;51(2):121-5
references.pmid: 21514213
references.citation: Trends Microbiol. 2004 Jun;12(6):279-87
references.pmid: 15165606
references.citation: J Food Prot. 2012 Jul;75(7):1292-302
references.pmid: 22980013
references.citation: J Clin Virol. 2010 Dec;49(4):254-7
references.pmid: 20864394
references.citation: J Virol Methods. 2000 Oct;90(1):69-78
references.pmid: 11011082
references.citation: J Pediatr Gastroenterol Nutr. 2005 Mar;40(3):328-33
references.pmid: 15735487
references.citation: J Clin Microbiol. 2000 Apr;38(4):1656-60
references.pmid: 10747162
references.citation: Virology. 1996 Mar 1;217(1):252-61
references.pmid: 8599210
references.citation: Proc Natl Acad Sci U S A. 2008 Jul 8;105(27):9175-80
references.pmid: 18599458
references.citation: Virology. 2006 Mar 15;346(2):312-23
references.pmid: 16343580
references.citation: Clin Infect Dis. 2007 Sep 1;45(5):534-40
references.pmid: 17682985
references.citation: Emerg Infect Dis. 2011 Jan;17(1):7-15
references.pmid: 21192848
references.citation: Curr Issues Mol Biol. 2011;13(2):51-76
references.pmid: 21502666
references.citation: J Clin Virol. 2013 Mar;56(3):194-8
references.pmid: 23177165