Isolation and characterization of conformation sensitive antierythropoietin monoclonal antibodies: effect of disulfide bonds and carbohydrate on recombinant human erythropoietin structure

Abstract

We have isolated and characterized three anti-recombinant human erythropoietin (rHuEPO) monoclonal antibodies (MoAbs) that recognize nonoverlapping epitopes on rHuEPO. Anti-EPO MoAb D11 neutralizes rHuEPO activity whereas MoAbs F12 and 9G8A do not. This suggests that D11 may bind to the rHuEPO active site. MoAbs F12 and D11 recognize conformation dependent epitopes whereas 9G8A does not. Immunoassays were developed for each monoclonal. The 9G8A immunoassay was novel and useful because immunoreactivity increased when rHuEPO was denatured. Disruption of disulfide bonds or removal of carbohydrate increased 9G8A immunoreactivity, which suggests that these elements are important for rHuEPO structure or stability.

Investigation of the effects of altered receptor binding activity on the clearance of erythropoiesis-stimulating proteins: Nonerythropoietin receptor-mediated pathways may play a major role

“…Serum levels of rHuEPO, NM294, NM340, darbepoetin alfa, PEG‐rHuEPO and PEG‐darbepoetin alfa) were measured with the Quantikine® enzyme‐linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). Serum concentrations of NM385 and PEG‐NM385 were determined with a modified ELISA assay using the EPO‐specific monoclonal antibody F12,34, 35 which recognizes different epitopes than the capture antibody provided with the Quantikine® ELISA kit; the secondary (detection) antibody was the same as for all other ELISA assays. All test agents were measured against their own standard curves.…”

“…The lower limit of quantification was 0.1 ng/mL for rHuEPO, 0.625 ng/mL for NM385, 2.5 ng/mL for PEG NM‐385, 1.25 ng/mL for PEG‐rHuEPO, 1.46 ng/mL for PEGylated darbepoetin alfa, and 0.1 ng/mL for all other ESAs. Endogenous mouse EPO showed minimal cross‐reactivity in the ELISA assays 34. However, a correction was made when necessary as described below.…”

Fine-structure epitope mapping of antierythropoietin monoclonal antibodies reveals a model of recombinant human erythropoietin structure

“…Mutations at amino acid residues 6, 7. 8, 10, 11,12,14,15,16,17,18, and 20 all gave equal or higher EPO concentrations measured with RIA-N, to that obtained with all other immunoassays except RIA-9GSA. However, compared to some other immunoassays a Ser' substitution reduced immunoreactivity over 10-fold and A d and Asn9 mutations reduced immunoreactivity approximately 30 to 35%.…”

Evaluation of AMGEN clone 9G8A anti‐Epo antibody for application in doping control

“…While clone AE7A5 has been widely used for the detection of rEPO misuse in doping control, clone 9G8A was developed by Amgen for internal research purposes only and has not been commercially available so far. [18,19] Aside from specificity issues, the sensitivity of both antibodies was also compared. High sensitivity is a prerequisite for detecting low amounts of rEPOs and other ESAs in human samples.…”