Plasma erythropoietin assay by a fetal mouse liver cell culture method with special reference to effective elimination of erythroid colony inhibitor(s) in plasma.
Methods for the elimination of an inhibitor(s) of erythroid colony formation from plasma were examined in an attempt to measure genuine plasma erythropoietin (Epo) activities with an erythroid colony-forming assay using fetal mouse liver cells. Acid-boiling-chloroform (ABC) treatment was concluded to be the best method because the plasma thus treated stimulated colony formation most and contained the least protein. The dose-response curve for the plasma was parallel to that for the standard Epo preparation. The "erythroid colony-stimulating activity" in the plasma was completely additive to that in the standard Epo, and appeared to be a relatively heat-stable and acid protein with an isoelectric point lower than 5.0. These results suggest that the activity in the plasma is identical to that in the standard Epo. Stability of the plasma Epo activity was dependent on storage temperature and enhanced by adding 1% bovine serum albumin (BSA). Average Epo titers for normal adult, full-term cord, and murine plasmas, all ABC-treated and with 1% BSA added, were 192.4, 184.5, and 150.6 mU/ml, respectively. These values were much higher than those measured by the in vivo standard polycythemic mouse assay.
D000818:Animals; D002478:Cells, Cultured; D003115:Colony-Stimulating Factors / Q000037:antagonists & inhibitors* / Q000097:blood; D003956:Dialysis; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000166:cytology*; D004921:Erythropoietin / Q000097:blood*; D005333:Fetus / Q000502:physiology; D006358:Hot Temperature; D006801:Humans; D008099:Liver / Q000166:cytology; D051379:Mice / Q000196:embryology; D013997:Time Factors
D003115:Colony-Stimulating Factors; D004921:Erythropoietin