CM Affi-Gel Blue chromatography of human urine: a simple one-step procedure for obtaining erythropoietin suitable for in vitro erythropoietic progenitor assays.
A method for both concentrating and purifying human urinary erythropoietin (Ep) using CM Affi-Gel Blue is described. We have found that up to 40 litres of urine can be processed on a 1 litre gel bed of this material. This gives a 25-50-fold purification of Ep with an apparent Ep recovery in excess of 100%. The high recovery of Ep is probably due, in part, to the removal of inhibitors present in the initial urine. By selecting urine that contains high levels of Ep (greater than 0.5 units/ml), it is possible with this method routinely to obtain preparations with specific activities of 100-300 units of Ep per mg protein. Such preparations are noninhibitory when assayed in either short-term suspension cultures or in longer-term methylcellulose cultures at concentrations up to 5-10 units/ml. Similar tests with these same bioassay systems have shown that other non-Ep stimulating factors (i.e. erythroblast enhancing factor (EEF), granulocyte/macrophage colony stimulating factor (GM-CSF) and burst promoting activity (BPA) ) are also not present at detectable levels. In this study we also show that the loss of biological activity which often occurs when partially purified Ep preparations are stored in solution is markedly reduced in the presence of either 1% bovine serum albumin or 0.1% sodium dodecyl sulphate.
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002847:Chromatography, Agarose / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D003114:Colony-Forming Units Assay; D004355:Drug Stability; D004356:Drug Storage; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects; D004921:Erythropoietin / Q000302:isolation & purification / Q000494:pharmacology / Q000652:urine*; D006098:Granulocytes / Q000166:cytology / Q000187:drug effects; D006801:Humans; D051379:Mice; D012685:Sepharose / Q000031:analogs & derivatives
C017842:cibacron blue-sepharose; D004921:Erythropoietin; D012685:Sepharose