A QUANTITATIVE BIOASSAY FOR ERYTHROPOIETIN, USING MOUSE BONE-MARROW

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  • 1 January 1981
    • journal article
    • research article
    • Vol. 97  (2) , 144-157
Abstract
A new in vitro assay for epo [erythropoietin] using the suspension culture-59Fe incorporation technique was devised based on the use of readily available adult mouse marrow cells. Particular attention was 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 Hb 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 was split into 2 parts separated by a centrifugation and cell wash step. Optimal conditions during the initial 24 h when cells were exposed to epo included the use of 20% FCS [fetal calf serum] (preselected for its ability to support eythroid colony formation in vitro), 0.1 mM .beta.-mercaptoethanol, and 2 .times. 106 marrow cells in a final culture volume of 0.5 ml. Optimal labeling during the subsequent 3 h exposure to [59Fe]ferrous citrate in serum-free medium required the addition of 50 .mu.g/ml transferrin. Substantial diminution of 59Fe incorporation was observed below 10 and above 150 .mu.g/ml. Optimal labeling iron concentrations were provided during the labeling period solely by 1 .mu.Ci [59Fe]ferrous citrate (10,000-25,000 .mu.Ci/mg Fe). 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 (23-25 h) made it possible to measure epo levels from 400 to 1 mU per 0.5 ml culture, with a corresponding variation in counts of 12-fold. 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 4-fold.

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