Polystyrene beads as an alternative support material for epitope identification of a prion-antibody interaction using proteolytic excision-mass spectrometry.
The binding epitope structure of a protein specifically recognized by an antibody provides key information to prevent and treat diseases with therapeutic antibodies and to develop antibody-based diagnostics. Epitope structures of antigens can be effectively identified by the proteolytic epitope excision-mass spectrometry (MS) method, which involves (1) immobilization of monoclonal or polyclonal antibodies, e.g., on N-hydroxysuccinimide-activated sepharose, (2) affinity binding of the antigen followed by limited proteolytic digestion of the immobilized immune complex, and (3) elution and mass spectrometric analysis of the remaining affinity-bound peptide(s). In the epitope analysis of recombinant cellular bovine prion protein (bPrP(C)) to a monoclonal antibody (mAb3E7), we found that epitope excision experiments resulted in extensive nonspecific binding of bPrP to a standard sepharose matrix employed. Here, we show that the use of amino-modified polystyrene beads with aldehyde functionality is an efficient alternative support for antibody immobilization, suitable for epitope excision-MS, with complete suppression of nonspecific bPrP binding.
Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland.
Tatiana
T
Pimenova
Lukas
L
Meier
Bernd
B
Roschitzki
Gabriela
G
Paraschiv
Michael
M
Przybylski
Renato
R
Zenobi
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011137:Polystyrenes / Q000737:chemistry*; D011328:Prions / Q000032:analysis* / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; D000939:Epitopes; D011137:Polystyrenes; D011328:Prions; D002918:Chymotrypsin