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.
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.
Mortezai
Naghmeh
N
Institute of Clinical Chemistry, University Medical Center Hamburg-Eppendorf, Martinistr 52, D-20246 Hamburg, Germany.
Harder
Sönke
S
Schnabel
Claudia
C
Moors
Eva
E
Gauly
Matthias
M
Schlüter
Hartmut
H
Wagener
Christoph
C
Buck
Friedrich
F
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
D015415:Biomarkers; D002272:Carcinoembryonic Antigen; D020543:Proteome; D014909:Wheat Germ Agglutinins