A quantitative bioassay for erythropoietin, using mouse bone marrow.

Abstract

A new in vitro assay for epo using the suspension culture-59Fe incorporation technique has been devised. The assay is based on the use of readily available adult mouse marrow cells. Particular attention has been given to defining conditions that would permit this assay to be applied to the evaluation of epo levels in a wide variety of test samples, including human sera, which contain factors in addition to epo that influence both the level of hemoglobin synthesis obtained and the extent of 59Fe incorporation. To eliminate variability in 59Fe uptake due to uncontrolled additions of transferrin or unlabeled iron from test samples, the assay has been split into two parts separated by a centrifugation and cell wash step. Optimal conditions during the initial 24 hr when cells were exposed to epo were found to include the use of 20% FCS (preselected for its ability to support erythroid colony formation in vitro), 0.1 mM beta-mercaptoethanol, and 2 X 10(6) marrow cells in a final culture volume of 0.5 ml. Optimal labeling during the subsequent 3 hr exposure to [59Fe]ferrous citrate in serum-free medium required the addition of 50 microgram/ml transferrin. Substantial diminution of 59Fe incorporation was observed below 10 and above 150 microgram/ml. Optimal labeling iron concentrations were provided during the labeling period solely by 1 muCi of [59Fe]ferrous citrate (10,000 to 25,000 muCi/mg of Fe). Thus no addition of unlabeled iron was necessary, and reduction of the specific activity of the added 59Fe by exogenous Fe from FCS or test samples was avoided. Careful choice of an appropriate prelabel incubation period (23 to 25 hr) made it possible to measure epo levels from 400 down to 1 mU per 0.5 ml culture, with a corresponding variation in counts of 12-fold. By comparison, the minimum epo level detectable with fetal liver cells under the same conditions was 10 mU/0.5 ml culture, and the maximum range in counts was less than fourfold.