title: Identification of stroke-associated-antigens via screening of recombinant proteins from the human expression cDNA library (SEREX).
abstract: BACKGROUND
  Because circulating antibodies against a variety of antigens have been detected in patients with coronary heart disease, carotid atherosclerosis and those who have suffered a stroke, it is suspected that immune response may be one of the mechanisms of atherogenesis The objective of this study is to identify novel antibodies in ischemic stroke patients by screening the expressed recombinant proteins using a human cDNA library (SEREX).
  
  
  METHODS
  To identify the candidate antigens, cDNA library was screened by SEREX using plasma from ten patients with ischemic stroke. Subsequently, via ELISA using recombinant proteins and synthetic peptides, the serum antibody levels were measured in two independent patient/healthy donor (HD) cohorts (142 and 78 in the 2nd screening and a validation cohort, respectively).
  
  
  RESULTS
  The initial screening resulted in the identification of six candidate antigens. Of these antigens, replication protein A2 (RPA2) was determined to be the antigen associated with stroke (P < 0.05) by ELISA with 2nd screening and validation cohort. Multifactorial logistic regression analysis showed that the increased levels of the RPA2 antibodies (RPA2-Abs) associated with stroke independent of other risk factors for stroke (P < 0.05). Receiver operating curve analysis demonstrated that the area under the curve from ELISA using GST fusion RPA2 and synthetic peptides (bRPA2-132) were 0.867 (95% CI: 0.798-0.936) and 0.971 (95% CI: 0.940-1.00), respectively. If the cut-off value of the bRPA2-132-Ab level was determined to be 0.334, the sensitivity and specificity of the antibody level as the diagnostic marker for stroke were 0.323 (95% CI: 0.209-0.453) and 1.00 (95% CI: 0.713-1.00), respectively.
  
  
  CONCLUSIONS
  SEREX identified RPA2 as the antigen associated with ischemic stroke and serum auto-antibodies against RPA2 elevates in stroke patients. RPA2-Abs could become a biomarker for the evaluation of ischemic stroke at risk.
authors.affiliation: Departments of Neurosurgery, Chiba Cardiovascular Center, Ichihara, Chiba, Japan. tmachida@ca2.so-net.ne.jp.
authors.forename: Toshio
authors.initials: T
authors.lastname: Machida
authors.affiliation: Department of Neurosurgery, Kameda Medical Center, Chiba, Japan. kubota.motoo@kameda.jp.
authors.forename: Motoo
authors.initials: M
authors.lastname: Kubota
authors.affiliation: Department of Neurological Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan. papez@xa2.so-net.ne.jp.
authors.forename: Eiichi
authors.initials: E
authors.lastname: Kobayashi
authors.affiliation: Department of Neurological Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan. iwadatey@faculty.chiba-u.jp.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Iwadate
authors.affiliation: Department of Neurological Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan. nsaeki@faculty.chiba-u.jp.
authors.forename: Naokatsu
authors.initials: N
authors.lastname: Saeki
authors.affiliation: Chiba Prefectural University of Health Sciences, Chiba, Japan. akiray@k2.dion.ne.jp.
authors.forename: Akira
authors.initials: A
authors.lastname: Yamaura
authors.affiliation: Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba, 260-8670, Japan. fnomura@faculty.chiba-u.jp.
authors.forename: Fumio
authors.initials: F
authors.lastname: Nomura
authors.affiliation: Department of Biochemistry and Genetics, Graduate School of Medicine, Chiba University, Chiba, Japan. mtak@faculty.chiba-u.jp.
authors.forename: Masaki
authors.initials: M
authors.lastname: Takiguchi
authors.affiliation: Department of Biochemistry and Genetics, Graduate School of Medicine, Chiba University, Chiba, Japan. hiwasa_takaki@faculty.chiba-u.jp.
authors.forename: Takaki
authors.initials: T
authors.lastname: Hiwasa
mesh_terms: D000368:Aged; D000906:Antibodies / Q000378:metabolism; D000941:Antigens / Q000378:metabolism*; D015153:Blotting, Western; D015331:Cohort Studies; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015723:Gene Library; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D010455:Peptides / Q000378:metabolism; D012372:ROC Curve; D011994:Recombinant Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D020521:Stroke / Q000276:immunology*
chemical_list: D000906:Antibodies; D000941:Antigens; D010455:Peptides; D011994:Recombinant Proteins