Characterization and use of monoclonal antibodies directed against human erythropoietin that recognize different antigenic determinants.
We have established four hybridoma cells that produce monoclonal antibodies (MoAbs) R2, R4, R6, and R12 directed toward recombinant human erythropoietin (rHuEPO). MoAbs R2, R4, and R6 bound to EPO with high affinities (kd = approximately 2, 4, and 1 nmol/L, respectively) but MoAb R12 had a low affinity (240 nmol/L). These antibodies inhibited the biological activity of rHuEPO and EPOs from humans, rats, mice, and rabbits. This inhibition was due to the blocking of EPO binding to the target cells. The fully deglycosylated rHuEPO bound to the MoAbs, indicating that they recognized peptide sequences of the antigen but not the carbohydrates attached to the antigen. An immunosorbent column with the immobilized MoAb R2 was effective for the rapid purification of EPO. MoAb R6 bound to EPO at a site(s) different from those to which other MoAbs bound. Based on this finding, a sensitive and rapid enzyme-linked immunosorbent assay of EPO, in which EPO was sandwiched between two MoAbs (R2 and R6), was developed. The assay measured plasma levels of EPO as low as 5 mU/mL within several hours.
Goto
M
M
Research Institute of Life Science, Snow Brand Mill Products, Tochigi, Japan.
Murakami
A
A
Akai
K
K
Kawanishi
G
G
Ueda
M
M
Chiba
H
H
Sasaki
R
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000502:physiology; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D004921:Erythropoietin / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D005260:Female; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D011956:Receptors, Cell Surface / Q000032:analysis; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin