In vitro bioassay of erythropoietic activity in serum using mouse spleen cells. The effect of heat inactivation on serum erythropoietin.
Untreated human serum is known to be toxic to in vitro assays for erythropoietin, including the mouse spleen cell assay system (MSCA). This phenomenon had previously been shown to be mediated by complement-dependent IgM heteroantibodies and can be overcome by heating the serum at 56 degrees C for 30 minutes. Using the MSCA, we have found that the toxic effect of serum could also be removed by treatment with a precipitating antibody against the C3c component of complement. The effects of the two methods of complement inactivation on the measurement of stimulatory activity in serum have been compared. For normal serum, the results after heat inactivation and antibody treatment were similar. In contrast, serum from a patient with aplastic anemia gave a result equivalent to 327 mU erythropoietin/ml after heat treatment, but after antibody treatment equivalent to 1,520 mU erythropoietin/ml. Gel permeation chromatography of unheated, heated, and antibody-treated sera showed that heating markedly reduced the activity of the erythropoietin peak. Seventy percent of the activity of partially purified urinary erythropoietin was lost during heating in the presence of normal serum. In addition, heating caused the appearance of high molecular weight compounds that are stimulatory in the MSCA. The level of this activity appeared to be directly related to the stimulatory activity of the unheated serum.
D000741:Anemia, Aplastic / Q000097:blood; D000818:Animals; D001681:Biological Assay; D001790:Blood Physiological Phenomena; D003165:Complement System Proteins / Q000494:pharmacology; D004920:Erythropoiesis / Q000187:drug effects*; D004921:Erythropoietin / Q000097:blood / Q000494:pharmacology*; D006358:Hot Temperature; D006801:Humans; D051379:Mice; D013154:Spleen / Q000187:drug effects
D004921:Erythropoietin; D003165:Complement System Proteins