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

Elliott, Steve; Lorenzini, Tony; Chang, David C.; Barzilay, Jack; Delorme, Evelyne; Giffin, James C.; Hesterberg, Lyndal K.

doi:10.1182/blood.v87.7.2702.bloodjournal8772702

Cited by 38 publications

(23 citation statements)

References 19 publications

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“…The 390 bp product was confirmed as having the erythropoietin sequence but lacking 57 bases. The missing sequence coded for 19 amino‐acids contributing to the third α‐helix, and a portion of the linking sequence to the fourth α‐helix, of the erythropoietin molecule (Elliot et al , 1996).…”

Section: Resultsmentioning

confidence: 99%

Phenotypic and molecular analysis of six human cell lines derived from patients with plasma cell dyscrasia

et al. 1999

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Summary. Cell lines RPMI 8226, JJN3, U266 B1, NCI-H929 (all EBV À ) and ARH77 and HS-Sultan (both EBV ) have been extensively characterized in this study. EBV À lines expressed the phenotype (CD138 À , CD19 , CD20 ) whereas EBV were (CD138 , CD19 À , CD20 À ). CD56 expression was restricted to EBV À cell lines, with the exception of U266 B1, whereas PCA-1 was strongly expressed on ®ve of the six cell lines. Only EBV cell lines bound peanut-agglutinin (PNA). However, all cell lines bound the lectin Jacalin that binds the same receptor as PNA, irrespective of the receptors sialylation status. By RT-PCR and direct sequencing of their IgH V/D/J domains, ARH77 was demonstrated to use the germline sequence VH 4 -34/dm1/JH6b, whereas no arrangement was demonstrated for RPMI 8226, suggesting IgH gene deletion or mutation. HLA class I and II antigens were detected using HLA typing on all cell lines warranting their use as suitable targets for HLA-restricted cytotoxic T cells. By sensitive RT-PCR, mRNA for IL-6, IL-6R and TNFb was found expressed in all cell lines. IL-1 mRNA expression was predominantly associated with the EBV phenotype. Although mRNA for IL-3 and GM-CSF was never detected, transcripts for c-kit ligand and, more commonly, its receptor were. Likewise GM-CSF, M-CSF and erythropoietin mRNA transcripts were detected in the majority of cell lines.

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Section: Resultsmentioning

confidence: 99%

Phenotypic and molecular analysis of six human cell lines derived from patients with plasma cell dyscrasia

et al. 1999

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“…In addition, immunoreactivity for EpoR has been demonstrated in the widespread population of tissue macrophages in the developing human fetus (Juul et al 1998). These findings have been questioned by Elliott et al (1996), who claimed that the C-20 (sc 695) anti-EpoR antibody used in these studies is a nonselective marker because it gives a false-positive signal in the absence of EpoR. In our present study, we have used a different antibody against EpoR and, alongside this, we have immunostained our samples with an anti-Epo antibody for Epo detection.…”

Section: Discussionmentioning

confidence: 91%

Mast cells and macrophages in duodenal mucosa of mice overexpressing erythropoietin

et al. 2009

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There is increasing evidence suggesting a wider biological role of erythropoietin (Epo) and Epo receptor (EpoR) not related to erythropoiesis, such as the detection of EpoR in other cells, i.e. polymorphonuclear leukocytes, megakaryocytes, endothelial, myocardial and neural cells. In this study, by using a mouse model (designated tg6) that constitutively overexpresses human Epo in an oxygen-independent manner, we have investigated mast cell and macrophage number and distribution in duodenal mucosa using immunohistochemical, morphometric and image analysis methods. The results showed that tryptase-positive mast cells and BM8-positive macrophages were more numerous in duodenal mucosa specimens of tg6 mice compared with wild-type mice. Moreover, whereas in wild-type specimens both mast cells and macrophages were generally scattered throughout the villus, in tg6 specimens they were aligned along the axis of the villus. Morphometric analysis confirms this observation, and the quantitative analysis of the spatial distribution of the cells in duodenal villi indicated that in both wild-type and tg6 groups the macrophage and mast cell distribution was characterized by significant deviations from randomness. In addition, an increased number of c-kit-positive cells have been identified in the villus axis of tg6 mice, indicating an expanded compartment of mast cell precursors in the intestinal mucosa of these animals. Finally, we have also demonstrated that in tg6 specimens the number of duodenal epithelial cells positive for Epo were significantly higher as compared to wild type. Overall, these data confirm that Epo, acting as a general stimulator of the hemopoietic compartment, is able to induce an expansion of two effectors of the immune response, mast cells and macrophages, in a specific peripheral site, the duodenal mucosa, in the tg6 mouse experimental model.

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“…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.…”

Section: Resultsmentioning

confidence: 99%

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

Reichel

Benetka

Lorenc

et al. 2016

Drug Testing and Analysis

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The two mouse monoclonal anti-erythropoietin (EPO) antibodies clone AE7A5 (generated by using a 26 amino acid N-terminal EPO-peptide) and 9G8A (developed by immunizing mice with full length human EPO) are both directed against linear epitopes at the N-terminus of EPO. While AE7A5 has been commercially available for many years, 9G8A was made for Amgen's internal research purposes. In the past, the commercial antibody was shown to cross-react with several proteins unrelated to EPO (e.g. E. coli thioredoxin reductase, zinc-α2-glycoprotein, S. cerevisiae enolase, human neuron-specific enolase, and human non-neuronal enolase). However, it displayed high sensitivity for detecting recombinant EPO (rEPO) misuse by athletes on Western blots. We evaluated the potential use of clone 9G8A for doping control purposes. While 9G8A showed lower sensitivity than AE7A5 (ca 45% on isoelectric focusing (IEF)-polyacrylamide gel electrophoresis (PAGE), ca 40% on sodium dodecyl sulfate (SDS)- and sarcosyl (SAR)-PAGE), non-specific binding of the five proteins was not observed. The cross-reactivity of AE7A5 can be overcome by immunoaffinity purification of EPO before electrophoresis and Western blotting. Similar to AE7A5, clone 9G8A is also suited for Western double-blotting. Copyright © 2016 John Wiley & Sons, Ltd.

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

Cited by 38 publications

References 19 publications

Phenotypic and molecular analysis of six human cell lines derived from patients with plasma cell dyscrasia

Phenotypic and molecular analysis of six human cell lines derived from patients with plasma cell dyscrasia

Mast cells and macrophages in duodenal mucosa of mice overexpressing erythropoietin

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

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