Raising monospecific antibodies by use of protein components prestained with Remazol Brilliant Blue and separated by disc electrophoresis on polyacrylamide gel.
We have reported (Clin Chem 29: 42-44, 1983) that prestaining with Remazol Brilliant Blue permits direct visualization of serum components on disc electrophoresis, and apparently purifies the proteins well. Here we have cut out the bands corresponding to the prestained albumin and transferrin after disc electrophoresis of normal human serum proteins, eluted some individual proteins into saline, and assessed their purity by immunoelectrophoresis and two-dimensional crossed immunoelectrophoresis against polyvalent antihuman serum. These two techniques indicated purity of these antigens. We inoculated rabbits with the eluates containing the pure antigens, and tested the resulting antibodies for monospecificity by immunoelectrophoresis, rocket electrophoresis, and single radial immunodiffusion. From the results we conclude that the antibodies raised against each component were monospecific, and that this is a simple, economical, rapid, and reliable method for obtaining a pure fraction of serum protein for use as an antigen.
D000328:Adult; D000880:Anthraquinones / Q000032:analysis; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D000941:Antigens / Q000276:immunology / Q000302:isolation & purification*; D004396:Coloring Agents; D004589:Electrophoresis, Disc; D006801:Humans; D005779:Immunodiffusion; D007123:Immunoelectrophoresis, Two-Dimensional; D012709:Serum Albumin / Q000276:immunology / Q000302:isolation & purification; D014168:Transferrin / Q000276:immunology / Q000302:isolation & purification
D000880:Anthraquinones; D000911:Antibodies, Monoclonal; D000941:Antigens; D004396:Coloring Agents; D012709:Serum Albumin; D014168:Transferrin; C024758:Remazol Brilliant Blue R