Inhibition of receptor binding and neutralization of bioactivity by anti-erythropoietin monoclonal antibodies.
We have generated four high affinity monoclonal antibodies (MoAbs) to recombinant human erythropoietin (EPO). All four MoAbs immunoprecipitate radioiodinated native EPO, and the concentrations of MoAbs required for maximum binding range from 10 nmol/L to 100 nmol/L. Two MoAbs, designated Group I MoAbs, bind to an epitope within the N-terminal 20 amino acids of EPO and also immunoprecipitate sodium dodecyl sulfate (SDS)-denatured EPO. Two other MoAbs (Group II MoAbs) do not immunoprecipitate SDS-denatured EPO and do not bind to any of the eight endo C fragments of EPO. We first used murine erythroleukemia (MEL) cells to test the MoAbs for inhibition of EPO-receptor binding. MEL cells, although unresponsive to EPO, express 760 high affinity receptors for EPO per cell (Kd = 0.24 nmol/L). To assay our MoAbs, MEL cells were grown as monolayers on fibronectin-coated Petri dishes and incubated at 4 degrees C with radioiodinated EPO. Group I MoAbs do not inhibit binding of radioiodinated EPO to the MEL EPO-receptor, but Group II MoAbs do inhibit binding in a dose-dependent manner. We next examined the neutralization of EPO bioactivity by our MoAbs, using EPO-dependent cell line. Only Group II MoAbs inhibit a newly developed EPO-dependent cell growth, demonstrating that inhibition of EPO-receptor binding correlates with neutralization of EPO bioactivity.
D'Andrea
A D
AD
Whitehead Institute for Biomedical Research, Cambridge, MA.
Szklut
P J
PJ
Lodish
H F
HF
Alderman
E M
EM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure; D004921:Erythropoietin / Q000276:immunology / Q000378:metabolism*; D004915:Leukemia, Erythroblastic, Acute / Q000097:blood / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D011233:Precipitin Tests; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017467:Receptors, Erythropoietin
D000911:Antibodies, Monoclonal; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D004921:Erythropoietin