title: Recombinant antibodies for the depletion of abundant proteins from human serum.
abstract: The identification of biomarkers from serum or plasma is often hindered by a few proteins present at high concentrations, which may obscure less abundant proteins. Ideal serum depletion strategies would be flexible as regards the proteins to be removed, and would feature the use of reagents with long shelf-lives. In this article, we describe a novel protein depletion methodology based on the incubation of serum samples with phage-derived recombinant antibody fragments, which are able to bind to staphylococcal Protein A, and which carry a C-terminal peptide tag capable of streptavidin binding. The resulting protein-antibody complexes can be removed by simultaneous capture on Protein A and/or streptavidin resin. The depletion methodology was exemplified by the isolation of recombinant human mAb fragments specific to abundant human serum Ags and by the simultaneous depletion of albumin, immunoglobulins, alpha2-macroglobulin, hemoglobin, transferrin and haptoglobin. The depleted serum samples were analyzed by 2-DE and by gel-free MS-based methodologies, confirming the efficiency and selectivity of the depletion process. The methodology presented is modular in nature, since several recombinant antibodies can be combined in a single depletion experiment. Furthermore, antibodies do not have to be covalently coupled to a solid support facilitating long-term storage.
authors.affiliation: Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, Switzerland.
authors.forename: Anna
authors.initials: A
authors.lastname: Ettorre
authors.forename: Christoph
authors.initials: C
authors.lastname: Rösli
authors.forename: Michela
authors.initials: M
authors.lastname: Silacci
authors.forename: Simon
authors.initials: S
authors.lastname: Brack
authors.forename: Gregor
authors.initials: G
authors.lastname: McCombie
authors.forename: Richard
authors.initials: R
authors.lastname: Knochenmuss
authors.forename: Giuliano
authors.initials: G
authors.lastname: Elia
authors.forename: Dario
authors.initials: D
authors.lastname: Neri
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D001798:Blood Proteins / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D040901:Proteomics; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000276:immunology
chemical_list: D000906:Antibodies; D001798:Blood Proteins; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A