Artificial antibodies for affinity chromatography of homologous proteins: application to blood clotting proteins.
A method to readily isolate antibodies that bind to only one member of a family of homologous proteins is described. A library of different single chain antibody fragments can be displayed on the surface of a bacteriophage vector. Individual antibodies from this library recognizing a particular protein from a family of homologous proteins can be readily isolated by a two-step affinity screening process. In the first step antibodies which bind specifically to the undesired proteins or to homologous regions of the proteins are removed. In the second step, those antibodies specifically recognizing the desired protein are then isolated. Using this procedure and starting with a naive antibody library, a single chain antibody fragment specific to the blood clotting protein, Protein C, which did not recognize either of the homologous proteins, Factor IX or Factor X, was isolated. Similarly an antibody specific to Factor IX, but not Factor X or Protein C, was also isolated. The isolated antibodies can be readily produced, purified, and affixed to sepharose beads for affinity chromatography of the blood clotting factors. One of the key advantages to this procedure over conventional monoclonal antibody isolation is that the antibodies are isolated and produced in vitro so a broad range of related proteins, toxins, viruses, or other products can be targeted.
Wu
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H
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, Baltimore, Maryland 21250, USA.
Goud
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GN
Sierks
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MR
D002846:Chromatography, Affinity; D005164:Factor IX / Q000276:immunology*; D005170:Factor X / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology* / Q000302:isolation & purification; D011486:Protein C / Q000276:immunology* / Q000302:isolation & purification
D007128:Immunoglobulin Fragments; D011486:Protein C; C055966:immunoglobulin Fv; D005164:Factor IX; D005170:Factor X