Isolation of Anti-Erythropoietin by Automated Immuno-Affinity FPLC using Recombinant EPO
Recently, the recombinant human glycoprotein, erythropoietin (EPO), has become available in a highly purified form. Erythropoietin of lower purity is normally isolated from urine. This preparation of EPO was used to immunize rabbits. The immunoglobulin-fraction (IgG) of the serum was isolated and further purified by immuno-affinity chromatography. For this purpose, a column was prepared by coupling 3 ug of recombinant EPO to 0.5 ml tresyl-activated SEPHAROSE 4B.The specific anti-EPO antibody fraction was isolated on this column by applying only small samples (400 ug of IgG) and running this column more than 800 times. This was only possible by completely automating the equipment (FPLC-system). One totally automated cycle of 80 runs lasted for about 4 days. After 800 runs the specific IgG fractions were pooled and concentrated by freeze-drying. The total amount of IgG in this pool was about 180 ug protein. Using this technique, it was possible to isolate larger quantities of immuno-affinity purified antibodies on only minute amounts of the recombinant protein. The same technique was applied to isolating immuno-affinty purified EPO on the specific IgG coupled to a similar matrix.The specific antibodies and the recombinant EPO were used in an enzyme-linked immunosorbent assay (dot-ELISA) to follow the purification of EPO. These purified proteins are being used to develop a new ELISA for EPO.
KeywordsAplastic Anemia Aplastic Anemia Patient Ammonium Sulfate Solution Saturated Ammonium Sulfate Solution Automate Cycle
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- 7.Mok M and Spivak JL(1982).Protease Activity in human urine erythropoietin preparations. Exp Hematol 10:300–306.Google Scholar