title: Avoiding false discovery in biomarker research.
abstract: Human tyrosine-protein phosphatase non-receptor type substrate 1α (SIRPA) is a surface marker identified in cardiomyocytes differentiated from human embryonic stem cells. Our objective was to determine if circulating SIRPA levels can serve as a biomarker of cardiac injury in children undergoing open heart surgery.
  
  
  
  Paired pre- and post-operative serum samples from 48 pediatric patients undergoing open heart surgery and from 6 pediatric patients undergoing non-cardiac surgery (controls) were tested for SIRPA protein levels using commercially available SIRPA ELISA kits from two manufacturers. Post-operative SIRPA concentrations were significantly higher in patients after cardiac surgery compared to non-cardiac surgery when tested using SIRPA ELISA kits from both manufacturers. To verify the identity of the protein detected, recombinant human SIRPA protein (rhSIRPA) was tested on both ELISA kits. The calibrator from both ELISA kits was analyzed by Western blot as well as by Mass Spectrometry (MS). Western blot analysis of calibrators from both kits did not identity SIRPA. MS analysis of calibrators from both ELISA kits identified several inflammatory markers and albumin but no SIRPA was detected.
  
  
  
  We conclude that commercially available ELISA kits for SIRPA give false-positive results. Verifying protein identity using robust protein characterization is critical to avoid false biomarker discovery when using commercial ELISA kits.
authors.affiliation: Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: Pranali
authors.initials: P
authors.lastname: Patel
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.|Ted Rogers Centre for Heart Research, Toronto, ON, Canada.
authors.forename: Uros
authors.initials: U
authors.lastname: Kuzmanov
authors.affiliation: Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada. seema.mital@sickkids.ca.|Department of Pediatrics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada. seema.mital@sickkids.ca.|Ted Rogers Centre for Heart Research, Toronto, ON, Canada. seema.mital@sickkids.ca.
authors.forename: Seema
authors.initials: S
authors.lastname: Mital
mesh_terms: D000943:Antigens, Differentiation / Q000097:blood* / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000097:blood*; D015153:Blotting, Western; D002138:Calibration; D016022:Case-Control Studies; D002648:Child; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D006335:Heart Injuries / Q000097:blood / Q000601:surgery; D006801:Humans; D013058:Mass Spectrometry; D011971:Receptors, Immunologic / Q000097:blood* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification
chemical_list: D000943:Antigens, Differentiation; D015415:Biomarkers; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C501920:SIRPA protein, human
keywords: Biomarker discovery; ELISA assay; Mass spectrometry; SIRPA; Validation