title: Immunoreactivity of anti-gelsolin antibodies: implications for biomarker validation.
abstract: BACKGROUND
  Proteomic-based discovery of biomarkers for disease has recently come under scrutiny for a variety of issues; one prominent issue is the lack of orthogonal validation for biomarkers following discovery. Validation by ELISA or Western blot requires the use of antibodies, which for many potential biomarkers are under-characterized and may lead to misleading or inconclusive results. Gelsolin is one such biomarker candidate in HIV-associated neurocognitive disorders.
  
  
  METHODS
  Samples from human (plasma and CSF), monkey (plasma), monocyte-derived macrophage (supernatants), and commercial gelsolin (recombinant and purified) were quantitated using Western blot assay and a variety of anti-gelsolin antibodies. Plasma and CSF was used for immunoaffinity purification of gelsolin which was identified in eight bands by tandem mass spectrometry.
  
  
  RESULTS
  Immunoreactivity of gelsolin within samples and between antibodies varied greatly. In several instances, multiple bands were identified (corresponding to different gelsolin forms) by one antibody, but not identified by another. Moreover, in some instances immunoreactivity depended on the source of gelsolin, e.g. plasma or CSF. Additionally, some smaller forms of gelsolin were identified by mass spectrometry but not by any antibody. Recombinant gelsolin was used as reference sample.
  
  
  CONCLUSIONS
  Orthogonal validation using specific monoclonal or polyclonal antibodies may reject biomarker candidates from further studies based on misleading or even false quantitation of those proteins, which circulate in various forms in body fluids.
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA.
authors.forename: Nicole
authors.initials: N
authors.lastname: Haverland
authors.forename: Gwënaël
authors.initials: G
authors.lastname: Pottiez
authors.forename: Jayme
authors.initials: J
authors.lastname: Wiederin
authors.forename: Pawel
authors.initials: P
authors.lastname: Ciborowski
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D018260:Gelsolin / Q000097:blood / Q000134:cerebrospinal fluid / Q000737:chemistry / Q000276:immunology*; D000882:Haplorhini; D006801:Humans; D013058:Mass Spectrometry; D015203:Reproducibility of Results; D017186:Titrimetry
chemical_list: D000906:Antibodies; D015415:Biomarkers; D018260:Gelsolin