title: Tandem affinity depletion: a combination of affinity fractionation and immunoaffinity depletion allows the detection of low-abundance components in the complex proteomes of body fluids.
abstract: Protein biomarker discovery in the low concentration range of human body fluids requires the enrichment of the proteins of interest. Here we report on a tandem affinity strategy: In the first step, we isolated a human plasma glyco-subproteome of healthy individuals by wheat germ agglutinin (WGA) lectin affinity chromatography. In the second step, the proteins of this subproteome were used to raise antibodies in llama (Lama glama). The heavy-chain fraction of the llama antibodies was used to deplete from the WGA lectin binding fraction all proteins normally found in human plasma. In this way, we selectively enriched the glycoprotein, CEA, a known cancer marker which had been spiked into normal plasma. As a proof of concept, we applied this method to the analysis of plasma sample from colon cancer patients. We could demonstrate the selective enrichment of CEA by a factor of 600-800.
authors.affiliation: Institute of Clinical Chemistry, University Medical Center Hamburg-Eppendorf, Martinistr 52, D-20246 Hamburg, Germany.
authors.forename: Naghmeh
authors.initials: N
authors.lastname: Mortezai
authors.forename: Sönke
authors.initials: S
authors.lastname: Harder
authors.forename: Claudia
authors.initials: C
authors.lastname: Schnabel
authors.forename: Eva
authors.initials: E
authors.lastname: Moors
authors.forename: Matthias
authors.initials: M
authors.lastname: Gauly
authors.forename: Hartmut
authors.initials: H
authors.lastname: Schlüter
authors.forename: Christoph
authors.initials: C
authors.lastname: Wagener
authors.forename: Friedrich
authors.initials: F
authors.lastname: Buck
mesh_terms: D000818:Animals; D000915:Antibody Affinity / Q000276:immunology; D015415:Biomarkers / Q000097:blood*; D015153:Blotting, Western; D002161:Camelids, New World / Q000276:immunology; D002272:Carcinoembryonic Antigen / Q000097:blood / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000097:blood; D006801:Humans; D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis* / Q000276:immunology; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D014909:Wheat Germ Agglutinins / Q000276:immunology
chemical_list: D015415:Biomarkers; D002272:Carcinoembryonic Antigen; D020543:Proteome; D014909:Wheat Germ Agglutinins