An effective and rapid method for functional characterization of immunoadsorbents using POROS beads and flow cytometry.
To facilitate the construction, functional characterization, and use of immunoadsorbents, we have developed a flow cytometry method that allows rapid assessment of large numbers of particle-bound antibodies. Protein G derivitized POROS beads were used to bind affinity-purified antibodies specific for synthetic peptides designed from human plasma proteins. The antibodies were covalently coupled to the beads and used to capture and release synthetic peptides that had been labeled at the C-terminus with the fluorochrome Alexa Fluor 488. Antibody coupling and specificity of antigen binding and release were measured by analysis of the POROS affinity beads by flow cytometry. The affinity-capture matrixes were also used through several antigen-binding and release cycles without loss of peptide binding efficiency. The ability to produce and characterize extremely small amounts of POROS affinity matrices will facilitate their use in protein microchemical procedures such as protein chip technology, monoclonal antibody screening and mass spectrometry, applications where analytes are limiting or present in low abundance in complex mixtures.
Anderson
N Leigh
NL
Plasma Proteome Institute, PO Box 53450, Washington, DC 20009-3450, USA.
Haines
Lee R
LR
Pearson
Terry W
TW
D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000941:Antigens / Q000737:chemistry; D002845:Chromatography / Q000379:methods; D004396:Coloring Agents / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D007164:Immunosorbents / Q000737:chemistry* / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits
C417561:Alexa 488 hydrazide; D000906:Antibodies; D000941:Antigens; D004396:Coloring Agents; D006834:Hydrazines; D007164:Immunosorbents; D010455:Peptides