Isolation of human erythropoietin with monoclonal antibodies.

Human erythropoietin was isolated from urine of aplastic anemic patients in a high yield with a simple purification procedure using an immunoadsorbent column of monoclonal antibodies and a Sephadex G-100 column.About 6 mg of erythropoietin was isolated from 700 liters of urine and the specific activity was estimated to be 81,600 unitslmg of protein with an in vivo 68Fe incorporation assay method, using starved rats.Activity measurement of the extracts from sliced gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Western blotting technique revealed heterogeneity of the isolated erythropoietin, which is probably caused by variable amounts of carbohydrates attached to the polypeptide chain.Thirty amino acids in the NH2-terminal portion of the isolated hormone were sequenced.Erythropoietin is a sialoglycoprotein which is believed to play an important role in regulating, by stimulating, erythropoiesis.Purification of erythropoietin from the urine of patients with aplastic anemia, using conventional purification procedures, has been reported (1, 2).However, laborious purification procedures with low yields and limited supplies of starting material have prevented us from obtaining amounts of pure erythropoietin suitable for studying its structure, mechanism of action, and metabolism.We have prepared a stable hybridoma clone that secretes monoclonal antibody against human urinary erythropoietin.'We describe here a very simple isolation procedure with a high yield of erythropoietin from human urine by the use of this monoclonal antibody. EXPERIMENTAL PROCEDURESMaterials-Erythropoietin (38,000 units/mg of protein with in vivo assay method) was purified from human urine by a combination of conventional methods, immunoadsorbent columns against contaminants and preparative SDS' polyacrylamide gel electrophoresis as a