title: Improved serological detection of rheumatoid arthritis: a highly antigenic mimotope of carbonic anhydrase III selected in a murine model by phage display.
abstract: BACKGROUND
  Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that affects around 1% of the human population worldwide. RA diagnosis can be difficult as there is no definitive test for its detection. Therefore, the aim of this study was to identify biomarkers that could be used for RA diagnosis.
  
  
  METHODS
  Sera from a collagen-induced arthritis mouse model were used to select potential biomarkers for RA diagnosis by phage display technology. In silico and in vitro analyses were performed to characterize and validate the selected peptides. Samples were classified into three groups: RA; two other immune-mediated rheumatic diseases (systemic lupus erythematosus (SLE) and ankylosing spondylitis (AS)); and healthy controls (HC). Enzyme-linked immunosorbent assay (ELISA) was carried out to determine antibody levels, and diagnostic parameters were determined by constructing receiver operating characteristic curves. Mass spectrometry and Western blot were performed to identify the putative autoantigen that was mimicked by a highly reactive mimotope.
  
  
  RESULTS
  After three rounds of selection, 14 clones were obtained and tested for immunoreactivity analysis against sera from RA and HC groups. The phage-fused peptide with the highest immunoreactivity (M12) was synthesized, and was able to efficiently discriminate RA patients from SLE, AS and HCs (p < 0.0001) by ELISA. The specificity and sensitivity of anti-M12 antibodies for RA diagnosis were 91 % and 84.3 %, respectively. The M12 peptide was identified as one that mimics a predicted antigenic site of the carbonic anhydrase III (CAIII) protein, a ubiquitous biomarker that has been identified in patients with other diseases.
  
  
  CONCLUSIONS
  M12 is the first peptide associated with the CAIII protein that may be used as an antigen for antibody detection to aid in RA diagnosis with high sensitivity and specificity.
authors.affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia, MG, Brazil. galber.araujo@gmail.com.|iMed.UL - Research Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal. galber.araujo@gmail.com.
authors.forename: Galber Rodrigues
authors.initials: GR
authors.lastname: Araujo
authors.affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia, MG, Brazil. emiliarezendev@gmail.com.
authors.forename: Emília Rezende
authors.initials: ER
authors.lastname: Vaz
authors.affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia, MG, Brazil. tiemebr@yahoo.com.br.
authors.forename: Patricia Tiemi
authors.initials: PT
authors.lastname: Fujimura
authors.affiliation: Rheumatology Research Unit, Institute of Molecular Medicine, Lisbon, Portugal. jecfonseca@gmail.com.|Rheumatology Department, Santa Maria Hospital, Lisbon Academic Medical Center, Lisbon, Portugal. jecfonseca@gmail.com.
authors.forename: João Eurico
authors.initials: JE
authors.lastname: Fonseca
authors.affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia, MG, Brazil. lucelia_bio@yahoo.com.br.
authors.forename: Lucélia Maria
authors.initials: LM
authors.lastname: de Lima
authors.affiliation: Rheumatology Research Unit, Institute of Molecular Medicine, Lisbon, Portugal. helenacanhao@gmail.com.|Rheumatology Department, Santa Maria Hospital, Lisbon Academic Medical Center, Lisbon, Portugal. helenacanhao@gmail.com.
authors.forename: Helena
authors.initials: H
authors.lastname: Canhão
authors.affiliation: Laboratory of Genetics and Molecular Cardiology, Heart Institute, University of São Paulo Medical School, São Paulo, SP, Brazil. venturini.gabriela@gmail.com.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Venturini
authors.affiliation: Fleury group, São Paulo, SP, Brazil. khmcardozo@gmail.com.
authors.forename: Karina Helena Morais
authors.initials: KH
authors.lastname: Cardozo
authors.affiliation: Fleury group, São Paulo, SP, Brazil. melechco@gmail.com.
authors.forename: Valdemir Melechco
authors.initials: VM
authors.lastname: Carvalho
authors.affiliation: Laboratory of Immunology and Molecular Biology, São Leopoldo Mandic Institute and Research Center, Campinas, SP, Brazil. marcelo.napimoga@gmail.com.
authors.forename: Marcelo Henrique
authors.initials: MH
authors.lastname: Napimoga
authors.affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia, MG, Brazil. lrgoulart@ufu.br.|Department of Medical Microbiology and Immunology, University of California Davis, Davis, CA, USA. lrgoulart@ufu.br.
authors.forename: Luiz Ricardo
authors.initials: LR
authors.lastname: Goulart
authors.affiliation: iMed.UL - Research Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal. joao.goncalves@ff.ul.pt.|IMM - Institute of Molecular Medicine, Lisbon, Portugal. joao.goncalves@ff.ul.pt.
authors.forename: João
authors.initials: J
authors.lastname: Gonçalves
authors.affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia, MG, Brazil. ueira@ingeb.ufu.br.
authors.forename: Carlos
authors.initials: C
authors.lastname: Ueira-Vieira
mesh_terms: D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D001172:Arthritis, Rheumatoid / Q000097:blood* / Q000175:diagnosis* / Q000235:genetics; D024403:Carbonic Anhydrase III / Q000097:blood* / Q000235:genetics; D061505:Cell Surface Display Techniques / Q000379:methods*; D004195:Disease Models, Animal; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008811:Mice, Inbred DBA; D008875:Middle Aged; D018716:Molecular Mimicry / Q000502:physiology*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary
chemical_list: D024403:Carbonic Anhydrase III