Generation and characterization of a neutralizing monoclonal antibody against erythroid cell stimulating factor

Erythroid cell stimulating factor (ESF) is present in mouse serum and has b een reported to function in concert with erythropoietin (EPO) in the format ion of erythroid cells in in vitro culture systems. We report here the gene ration and characterization of a monoclonal antibody (MAb) directed against ESF, with potent anti-ESF-neutralizing activity. A hybridoma-producing MAb to ESF was selected following enzyme-linked immunosorbent assay (ELISA)-ba sed screening of 270 colonies obtained from a fusion of immunized mouse spl enocytes with NS1 myeloma cells. Western blot analyses of mouse serum using this antibody specifically detected a single protein (approximate molecula r weight of 60 kDa and 120 kDa, under reducing and nonreducing conditions, respectively) corresponding to ESF, with no reactivity to EPO. Furthermore, this MAb demonstrated reactivity to a protein similar in molecular mass, a cross species, showing reactivity in sera obtained from human, horse, goat, guinea pig, rabbit, and rat. Immuno-chemical characterization demonstrated this antibody to be of IgG3 isotype, bearing kappa light chains. Injection of this monoclonal anti-ESF antibody to exhypoxic polycythemic mice at 6 a nd 24 h after EPO injection significantly reduced Fe-59 incorporation into red blood cells, demonstrating its ability to neutralize in vivo erythropoi esis in our mouse model system. Thus, this novel erythroid cell-specific MA b will be an invaluable tool for further delineating the physiological role of ESF in in vivo erythropoiesis.