SERUM ERYTHROPOIETIN BIOASSAY USING A NEW CELL LINE

WE PLAN TO EXPLOIT THE ISOLATION OF A CELL LINE FROM ERYTHROLEUKEMIC MURINE TUMORS WHICH IS HIGHLY SENSITIVE LINE FROM ERYTHROLEUKEMIC MURINE TUMORS WHICH IS HIGHLY SENSITIVE TO ERYTHROPOIETIN. WE WILL UTILIZE THIS LINE TO DEVELOP A SENSITIVE AND SPECIFIC BIOASSAY FOR ERYTHROPOIETIN IN HUMAN SERUM. OUR PRELIMINARY WORK HAS SHOWN THAT WE HAVE ISOLATED A LINE WHICH IS SENSITIVE TO VERY LOW LEVELS OF ERYTHROPOIETIN. THIS LINE, HC24 HAS NOW BEEN CARRIED CONTINUOUSLY FOR MORE THAN 120 PASSAGES OVER AN 18 MONTH PERIOD. IT DOES NOT APPEAR TO BE SENSITIVE TO A VARIETY OF OTHER GROWTH FACTORS INCLUDING IL-1, IL-2, IL-3, GM-CSF OR BY WORD OF MOUTH (DR. S. RUSCETTI) IL-4, IL-5, IL-6 AND G-CSF. THE DESIGN OF THIS SPECIFIC STUDY IS TO ISOLATE AND CHARACTERIZE STABLE, HIGHLY EPO SENSITIVE CLONAL CELL LINES FROM THE PARENTERAL HC LINES. WE WILL USE A TRITIATED THYMIDINE UPTAKE PROCEDURE DESCRIBED BY KRYSTAL TO ASSAY RESPONSE TO ERYTHROPOIETIN BUT WILL ALSO EVALUATE THE POSSIBILITY OF USING AN MTT SUBSTRATE ASSAY TO REPLACE TRITIATED THYMIDINE. WE WILL COMPARE THESE METHODS AND WILL DETERMINE THE FEASIBILITY OF ASSAYING BIOLOGIC ACTION OF EPO IN-VITRO IN THE PRESENCE OF HUMAN SERUM. WE WILL LOOK FOR THE EFFECTS OF HUMAN SERUM ON INHIBITING CELLS AND WILL USE ANTIBODY DEPLETION OF SAMPLES REMOVING EPO TO SEE WHETHER OR NOT ACTIVITY IS REMOVED. WE WILL LOOK FOR THE EFFECTS OF HUMAN SERUM ON QUANTITATING EPO AND ANALYZE THE ABILITY TO DETECT VERY LOW LEVELS IN VERY SMALL VOLUMES.