anti-EPO Antibodies Research Articles

The (re)challenging question of erythropoiesis-stimulating agents inducing pure red cell aplasia

The introduction of recombinant human erythropoietin (EPO) into routine clinical practice in the late 1980s heralded a new dawn for the treatment of anaemia in chronic kidney disease (CKD). Apart from the financial burden, limiting its use in the developing world, the early sideeffect profile was mild. It has been shown that full correction of anaemia, requiring high doses of EPO, does not confer mortality benefit to patients suffering from CKD [1,2], and is potentially detrimental to survival [1] even in haemodialysis patients with cardiac disease; the administration of EPO to raise haematocrit to 42% is not recommended [3]. Pure red cell aplasia (PRCA), secondary to EPO, will form the basis of this review. This idiosyncratic immunological reaction involves the generation of anti-EPO antibodies (Abs) resulting in significant anaemia. The mechanisms governing this immunological response remain only partially understood. Whilst PRCA is infrequent, the disease is severe, and the aggressive treatment requirements present a significant complication to patients and a therapeutic conundrum to the treating clinician.

Differential Neuroprotective Properties of Endogenous and Exogenous Erythropoietin in a Mouse Model of Traumatic Brain Injury

Both heat acclimation (HA) and post-injury treatment with recombinant human erythropoietin (Epo, rhEpo, exogenous Epo) are neuroprotective against traumatic brain injury (TBI). Our previous data demonstrated that HA-induced neuroprotection includes improved functional recovery and reduced cerebral edema formation. Additionally, in earlier Western-blot analyses, we found that HA mice display increased expression of the specific erythropoietin receptor (EpoR) and of hypoxia-inducible factor-1 alpha (HIF-1 alpha), the inducible subunit of the transcription factor, which regulates Epo gene expression, but not of Epo itself. In light of this, the aim of the current study was threefold: (1) to assess Epo expression in the trauma area and hippocampus following HA, rhEpo administration, or combined HA-rhEpo treatment, using immunohistochemical methods that offer enhanced anatomical resolution; (2) to examine the effects of endogenous and exogenous Epo on edema formation in normothermic (NT) mice; and (3) to evaluate the effects of exogenous Epo administration on neuroprotective outcome measures in HA animals. HA induced enhanced expression of endogenous Epo in the trauma area and the hippocampus. Treatment with anti-Epo antibody given to NT mice increased edema formation, whereas rhEpo induced no beneficial effect. Cognitive performance testing and immunohistochemical findings reinforced HA and rhEpo as separate protective interventions but showed no advantage to combining the two strategies. We therefore suggest that HA-induced neuroprotection is shaped by pre-existing mediators but cannot be modified by post-injury treatment aimed at increasing the levels of neuroprotective agents.

New alternatives for erythropoietin therapy in chronic renal failure

Abstract Erythropoietin (EPO) is one of the main cytokines involved in the regulation of erythropoiesis. The main site of EPO production are the kidneys. An altered EPO production leads to pathological conditions such as anemia and polycythaemia. Due to the progressive loss of renal peritubular cells, patients with chronic kidney disease (CKD) have low EPO plasma levels. This decreases erythron stimulation with the direct consequence of developing anemia. Before the introduction in the clinical practice of rHuEpo, in the late 1980s, the only solution for treating this type of anemia were blood transfusions and anabolic steroids. Even rHuEpo has proven to be safe and effective for treatment of anemias, there are some concerns about its cost, the need for frequent parenteral administration, and development of anti-EPO antibodies. These inconveniences prompted the search for novel erythropoiesis stimulating agents. Different strategies lead to isolation or chemical synthesis of such agents as darbepoetin alfa and EPO mimetics. In this review, we present some general aspects of EPO biology, with emphasis on chronic renal failure, and expose some of the alternatives to EPO used for anemia correction.

Characterization of erythropoietin receptor and erythropoietin expression and function in human ovarian cancer cells

The identification of erythropoietin receptors (EpoR) on cancer cells has caused concern, since it implies the possibility that treatment of cancer patients with erythropoietin (Epo) and related agents with demonstrable antiapoptotic activity could enhance cancer growth and progression. However, the function and even the validity of the identification of these receptors have been called into question. We now report the characterization of EpoR and Epo expression by 4 human ovarian cancer cell lines: A2780, CaOV, SKOV and OVCAR-3. Using semiquantitative RT-PCR, restriction digestion of the PCR products and DNA sequence analysis, we determined that each of the lines expresses the EpoR and Epo at the mRNA level. A2780 cells were the highest expressers of both genes. We demonstrated EpoR protein both by western blotting and by immunofluorescence and biologically active Epo protein by quantitative in vitro bioassay. The EpoR on A2780 cells was shown to be functional, since Epo stimulation resulted in phosphorylation of Erk1/2, an important EpoR mitogenic signaling intermediate. None of the cell lines exhibited a growth response in culture to exogenous Epo. However, addition of a neutralizing anti-Epo antibody to A2780 cells resulted in partial growth inhibition that was reversed by the addition of excess Epo, providing evidence for an autocrine/paracrine mechanism of growth enhancement in these cells.

Biological Significance of Erythropoietin and Erythropoietin Receptor Expression by Human Ovarian Cancer Cells.

Erythropoietin (Epo) is the primary regulator of erythroid cell proliferation and differentiation mediated through its specific binding to the Epo receptor (EpoR). Epo also is known to have non-hematopoietic actions, including the promotion of wound healing, tissue protection and others. Accumulating evidence demonstrating both Epo and EpoR expression in many types of human cancers has raised concern about the clinical use of Epo and other erythropoiesis stimulating agents (ESAs) in cancer-related anemia, since the presence of functional EpoR on the tumor cells suggests the potential for an adverse effect of Epo treatment by enhancing cancer growth and progression. Previously, we demonstrated that A2780 ovarian cancer cells express the EpoR gene and that long-term Epo treatment of these cells renders them resistant to paclitaxel. We now report the characterization and biological significance of Epo and EpoR expression by four ovarian cancer cell lines. Using semi-quantitative RT-PCR, restriction digestion of the PCR products and DNA sequence analysis, we demonstrated that A2780, CaOV, SKOV, and OVCAR-3 ovarian cancer cell lines examined express Epo and EpoR at the mRNA level. We demonstrated EpoR protein expression both by western blotting and by immunofluorescence, and demonstrated biologically active Epo protein expression by quantitative in vitro bioassay of the cell culture supernatants. The EpoR on A2780 cells are functional, since Epo stimulation of the cells resulted in a time-dependent 5-fold increase in phosphorylation of Erk1/2 that reached a peak in 10 min and then returned to the basal level. None of the cell lines exhibited a short-term (3-day) growth response in culture to exogenous Epo. However, addition of a neutralizing anti-Epo antibody to the cell culture resulted in partial growth inhibition of A2780 cells that was reversed by addition of excess Epo, consistent with an autocrine/paracrine mechanism of Epo growth enhancement of these cells. We also found that long-term Epo treatment of A2780 cells resulted in the development of a phenotype exhibiting enhanced Epo signaling, evidenced by a 20-fold increase in phosphorylation levels of Erk1/2. This phenotype was sustained even after the removal of Epo, suggesting a mechanism underlying the paclitaxel resistance developed after long-term Epo treatment. Our findings have implications for the clinical use of recombinant human Epo and other ESAs to correct and/or prevent anemia in ovarian and, potentially, other cancer patients. They also suggest that endogenous Epo and the EpoR may serve as novel therapeutic targets in the treatment of cancer patients.

Increased expression of erythropoietin receptor in human pterygial tissues

Erythropoietin (Epo) induces physiological activities such as cell proliferation, migration, and angiogenesis in Epo receptor (EpoR)-expressing vascular endothelial and tumor cells. Recently, it has been demonstrated that growth factor-independent proliferation is frequently observed during the cell transformation process. Pterygium is a fibrovascular proliferating tissue that includes transformed cells. The aim of this study was to examine the localization of Epo and EpoR proteins in human pterygial tissues. Eleven samples including nine pterygia and two normal bulbar conjunctivas, which were surgically excised, were studied. Formalin-fixed, paraffin-embedded tissue sections were constructed and then were examined by immunohistochemistry with anti-Epo and EpoR antibodies. Cytoplasmic immunoreactivity for EpoR was heterogeneously detected in basal and suprabasal cells of the pterygium epithelium. In the pterygium stroma, a variety of endothelial cells forming vascular cavities showed cytolasmic immunoreactivity for EpoR. In normal conjunctival epithelium, a few basal cells showed a weak homogeneous immunoreactivity for EpoR in the cytoplasm. The number of EpoR-expressing epithelial cells was much higher in the pterygium compared to the normal conjunctiva. EpoR expression was marginally detected in stromal microvessels of the normal conjunctiva. Immunoreactivity for Epo was not noted in pterygium epithelium and stroma, and in normal conjunctiva. These results suggest that the Epo-independent EpoR-signaling pathway plays a potential role in cell proliferation and angiogenesis in human pterygium.

Testing for recombinant erythropoietin

Erythropoietin (Epo) is a glycoprotein hormone that promotes the production of red blood cells. Recombinant human Epo (rhEpo) is illicitly used to improve performance in endurance sports. Doping in sports is discouraged by the screening of athletes for rhEpo. Both direct tests (indicating the presence of exogeneous Epo isoforms) and indirect tests (indicating hematological changes induced by exogenous Epo administration) can be used for Epo detection. At present, the test adopted by the World Anti Doping Agency is based on a combination of isoelectric focusing and double immunoblotting, and distinguishes between endogenous and rhEpo. However, the adopted monoclonal anti-Epo antibodies are not monospecific. Therefore, the test can occasionally lead to the false-positive detection of rhEpo (epoetin-beta) in post-exercise, protein-rich urine, or in case of contamination of the sample with microorganisms. An improved preanalytical care may counteract a lot of these problems. Adaptation of the criteria may be helpful to further refine direct Epo testing. Indirect tests have the disadvantage that they require blood instead of urine samples, but they can be applied to detect a broader range of performance improving techniques which are illicitly used in sports.

L-Arginine Administration Reverses Anemia Associated with Renal Disease

Recombinant human erythropoietin (rhEpo) has proved to be remarkably safe and effective for the treatment of anemia. Despite the use of rhEpo, concerns about its cost, the need for frequent parenteral administration, and the development of anti-Epo antibodies have prompted the development of improved agents to rescue anemia. Patients with anemia associated with renal disease are usually treated by intravenous or subcutaneous rhEpo administration; however, some patients do not respond well to rhEpo, because of the presence of Epo antibody or other unknown reasons. A new, orally administered drug is needed as an economical and effective method to treat such patients. We administered 1.3 g/day of L-arginine to 8 elderly patients with anemia associated with renal disease. All 8 patients responded to the treatment with increases in hemoglobin levels. Six of the patients showed improved renal function. There were no significant adverse effects. Our data show that oral administration of 1.3 g/day of L-arginine significantly improves Epo production and reverses anemia without adverse effects in elderly patients who have anemia associated with renal disease and are in the predialysis state of chronic renal failure.

Hematide is immunologically distinct from erythropoietin and corrects anemia induced by antierythropoietin antibodies in a rat pure red cell aplasia model

To evaluate the potential of Hematide, a PEGylated, synthetic peptide-based erythropoiesis-stimulating agent that is in clinical development for the treatment of anemia associated with chronic kidney disease and cancer, to correct antierythropoietin antibody-associated pure red cell aplasia (PRCA). The binding of anti-Hematide antibodies (mouse, rabbit, and monkey) to recombinant human erythropoietin (rHuEPO) and of anti-rHuEPO antibodies (mouse, goat, rat, and human) to Hematide were evaluated. An anti-EPO antibody-mediated anemia rat model was developed by subcutaneously administering rHuEPO to rats three times weekly for 4 weeks. Sixty percent of the animals developed PRCA as characterized by severe anemia, reduced reticulocytes, anti-EPO antibodies, and limited bone marrow erythroid precursors. The effect of Hematide administration on the PRCA rats was evaluated. Antibodies to EPO do not cross react with Hematide and, conversely, antibodies to Hematide do not cross react with EPO. Hematide corrected antibody-induced anemia in a rat PRCA model. The data support the potential of Hematide to correct anti-EPO antibody-associated PRCA in humans. In addition, the data suggest a negligible risk for development of anti-EPO antibody-induced PRCA secondary to Hematide administration.

Erythropoietin Blockade Inhibits the Induction of Tumor Angiogenesis and Progression

BackgroundThe induction of tumor angiogenesis, a pathologic process critical for tumor progression, is mediated by multiple regulatory factors released by tumor and host cells. We investigated the role of the hematopoietic cytokine erythropoietin as an angiogenic factor that modulates tumor progression.Methodology/Principal FindingsFluorescently-labeled rodent mammary carcinoma cells were injected into dorsal skin-fold window chambers in mice, an angiogenesis model that allows direct, non-invasive, serial visualization and real-time assessment of tumor cells and neovascularization simultaneously using intravital microscopy and computerized image analysis during the initial stages of tumorigenesis. Erythropoietin or its antagonist proteins were co-injected with tumor cells into window chambers. In vivo growth of cells engineered to stably express a constitutively active erythropoietin receptor EPOR-R129C or the erythropoietin antagonist R103A-EPO were analyzed in window chambers and in the mammary fat pads of athymic nude mice. Co-injection of erythropoietin with tumor cells or expression of EPOR-R129C in tumor cells significantly stimulated tumor neovascularization and growth in window chambers. Co-injection of erythropoietin antagonist proteins (soluble EPOR or anti-EPO antibody) with tumor cells or stable expression of antagonist R103A-EPO protein secreted from tumor cells inhibited angiogenesis and impaired tumor growth. In orthotopic tumor xenograft studies, EPOR-R129C expression significantly promoted tumor growth associated with increased expression of Ki67 proliferation antigen, enhanced microvessel density, decreased tumor hypoxia, and increased phosphorylation of extracellular-regulated kinases ERK1/2. R103A-EPO antagonist expression in mammary carcinoma cells was associated with near-complete disruption of primary tumor formation in the mammary fat pad.Conclusions/SignificanceThese data indicate that erythropoietin is an important angiogenic factor that regulates the induction of tumor cell-induced neovascularization and growth during the initial stages of tumorigenesis. The suppression of tumor angiogenesis and progression by erythropoietin blockade suggests that erythropoietin may constitute a potential target for the therapeutic modulation of angiogenesis in cancer.

Anti-EPO and anti-NESP antibodies raised against synthetic peptides that reproduce the minimal amino acid sequence differences between EPO and NESP

Erythropoietin (EPO) is a hormone that regulates red blood cell production. Recombinant human EPO (rHuEPO) and NESP (novel erythropoiesis stimulating protein) have been produced for therapeutic purposes and also to improve sports performance. The primary sequences of rHuEPO and NESP differ by just five amino acids. Due to the high homology, no antibodies that are able to discriminate between both molecules have been obtained until now. The aim of the present work was to design synthetic peptides corresponding to the sequence that differs between EPO and NESP (87-90aa), that can then be used as immunogens to develop specific rabbit polyclonal antibodies for selectively detecting EPO and NESP. Three peptides were synthesized: EPO (81-95), NESP (81-95), and NESP (86-104), and these were coupled to KLH and OVA for immunization and screening purposes, respectively. The sera obtained were tested by ELISA on synthetic peptide-OVA conjugates and purified by immunoaffinity chromatography against the corresponding synthetic peptide. The specific purified antibodies were characterized by ELISA, SDS-PAGE, and isoelectric focusing, followed by western blot. Antisera raised against EPO (81-95) recognized rHuEPO but not NESP. In contrast, anti-NESP (84-106) sera gave a specific anti-NESP response only after immunoaffinity purification on a NESP (86-91) column. An efficient strategy for generating specific antibodies against EPO and NESP can be achieved by selecting suitable synthetic peptides. The antibodies obtained are able to differentiate between rHuEPO and NESP, and may be particularly useful for screening purposes in both therapeutic and antidoping contexts.

Erythropoietin/erythropoietin‐receptor system is involved in angiogenesis in human hepatocellular carcinoma

Ribatti D, Marzullo A, Gentile A, Longo V, Nico B, Vacca A & Dammacco F (2007) Histopathology 50, 591–596 Erythropoietin/erythropoietin-receptor system is involved in angiogenesis in human hepatocellular carcinomaAimsTo correlate microvascular density and erythropoietin (Epo)/Epo-receptor (EpoR) expression in endothelial and tumour cells with histopathological type in hepatocellular carcinoma (HCC).Methods and resultsSpecimens of primary HCC obtained from 50 patients who had undergone curative hepatectomy were investigated immunohistochemically by using anti-CD31, anti-Epo and anti-EpoR antibodies. Poorly differentiated HCC had a higher degree of vascularization than other stages and Epo/EpoR expression in both tumour and endothelial cells increased in parallel with grade of malignancy and was highly correlated with the extent of angiogenesis.ConclusionsEpo/EpoR levels correlate with angiogenesis and progression of patients with HCC and these findings suggest the presence of a loop in the Epo/EpoR system, i.e. Epo is secreted by hepatic tumour cells and it affects vascular endothelial cells via its receptors and promotes angiogenesis in a paracrine manner. It is thus suggested that Epo is an important factor in hepatic tumour angiogenesis. Understanding the mechanisms of HCC angiogenesis provides a basis for a rational approach to the development of antiangiogenic therapy in patients with hepatic cancer.

Erythropoietin/erythropoietin receptor system is involved in angiogenesis in human neuroblastoma

Ribatti D, Poliani P L, Longo V, Mangieri D, Nico B & Vacca A (2007) Histopathology50, 636–641 Erythropoietin/erythropoietin receptor system is involved in angiogenesis in human neuroblastomaAimsPrevious studies have shown that increased vascularity is associated with tumour progression in human neuroblastoma (NB). The involvement of erythropoietin (Epo) in tumour angiogenesis has also been reported. The aim of this study was to correlate microvascular density and Epo/Epo-receptor (EpoR) expression in endothelial and tumour cells to the clinical stage of NB.Methods and resultsSpecimens of NB obtained from 20 patients were investigated immunohistochemically by using anti-CD31, anti-Epo and anti-EpoR antibodies. The extent of angiogenesis was found to be up-regulated in advanced disease. In keeping with this observation, Epo/EpoR expression in tumour and endothelial cells, respectively, was also highly correlated with the extent of angiogenesis and higher clinical stage.ConclusionsThe correlation of Epo/EpoR expression with angiogenesis and tumour progression suggests the presence of a loop in the Epo–EpoR system. Epo is secreted by tumour cells and affects vascular endothelial cells via its receptor, promoting tumour angiogenesis in a paracrine manner. Data suggest that Epo represents an important mediator in NB angiogenesis. Understanding the mechanisms of NB angiogenesis provides the basis for a rational approach to the development of antiangiogenic therapy in patients affected by NB.

Antibody-mediated acquired pure red cell aplasia (PRCA) after treatment with darbepoetin

Recombinant erythropoietin (EPO) is used successfully as treatment for anaemia in chronic kidney disease (CKD), to maintain stable haemoglobin levels, avoiding the need for multiple transfusions. From 1998, epoetin formulations, in particular epoetin alfa (Eprex , Ortho Biologics LLC), have been associated with a substantial increase in the incidence of antibody (Ab)-mediated pure red cell aplasia (PRCA) [1]. PRCA is a rare immunopathological disorder characterized by a severe reticulocytopenia, the absence of marrow erythroid precursors, normal granulocytic and megakaryocytic precursors, and the identification of circulating antibodies against EPO [2]. Patients typically develop an abrupt onset of severe transfusiondependent anaemia. Consequently, EPO must be withdrawn and therapy initiated to decrease the production of the anti-EPO antibodies. Darbepoetin alfa (Aranesp , Amgen Inc., CA) is a second generation erythropoiesis stimulating agent with two extra carbohydrate chains and eight extra sialic acid residues as compared with epoetin alfa. It is suggested that the modifications to this molecule may make it more resistant to antibody development. There is only a single published case report of PRCA related to the exclusive use of darbepoetin alfa [3]; however, communication with Amgen Australia Pty Ltd indicate about four cases on their database. In a sole published case of PRCA due to darbepoetin alfa, the patient did not respond to treatment with steroids alone, intravenous immunoglobulin, cyclosporin or combination prednisolone and cyclophosphamide [3]. We report a second case of PRCA related to the exclusive use of darbepoetin alfa, where the patient responded to the first line treatment of oral cyclophosphamide and prednisolone, had non-neutralizing anti-EPO and anti-darbepoetin antibodies on bioassay, and died from severe pulmonary infection.

Role of Erythropoietin as an Angiogenic Factor and Target in Cancer.

Erythropoietin is a pleiotropic cytokine with diverse non-hematopoietic functions. Epo and its receptor EpoR have been implicated in the physiologic angiogenesis that occurs in the mouse embryo, female genital tract and during wound healing. Epo is involved in the pathologic angiogenesis of diabetic retinopathy but the role of Epo in tumor angiogenesis, a pathologic process that is essential for tumor progression, is not established. We investigated the hypothesis that Epo may play an important role in tumor cell-induced angiogenesis and progression. Fluorescently-labeled rodent mammary carcinoma cells were implanted in dorsal skin-fold window chambers in nu/nu mice, a model that allows direct, non-invasive, serial visualization and real-time assessment of tumor cells and neovascularization simultaneously during the initial stages of tumor formation. Tumor angiogenesis (vascular length density, VLD) and growth (tumor area) were measured serially over 8 days using intravital microscopy and computerized image analysis. Co-injection of Epo with implanted tumor cells significantly increased VLD by 78% compared to vehicle-injected controls (p<0.001 by repeated measures ANOVA and Bonferroni test, n=8 mice/group). This early proangiogenic effect was associated with significant stimulation of tumor growth by 66% (p<0.001). Implantation of R3230-GFP cells engineered to stably express a constitutively active EpoR mutant (R129C) resulted in significant stimulation of neovascularization by 76% (p<0.001, n=7) and increased tumor growth by 79% compared to empty vector-transfected control cells (p<0.001). To target endogenous Epo function in window chambers, recombinant soluble EpoR (sEpoR) or a neutralizing anti-Epo monoclonal antibody (mAb) were co-injected at the time of tumor cell implantation. By day 8, marked inhibition of neovascularization was observed in response to treatment with sEpoR (44% reduction) or mAb (47% reduction) compared to vehicle-injected controls (p<0.001, n=7). This anti-angiogenic effect was associated with significant decrease in tumor size by 37% (sEpoR) and 39% (mAb), respectively (p<0.001). Stable expression of a secreted Epo antagonist (Epo-R103A) protein in tumor cells was associated with a remarkable anti-angiogenic effect with 50% reduction in VLD (p<0.001, n=7) and near complete disappearance of tumor cells by day 8 compared to controls (p<0.001). Analysis of two independent single cell clones of each transfected cell line showed similar results. All transfected cell lines exhibited similar in vitro growth characteristics and cell cycle profile. To further assess in vivo tumor growth, cells were implanted orthotopically in the mammary fat pad of female nu/nu mice (10 animals/group). EpoR-R129C expression was associated with significantly increased tumor volume (531±41 mm3, n=10) compared to vector-transfected cells (232±46 mm3, n=7). EpoR-R129C tumors exhibited significantly increased numbers of positive cells for proliferation marker Ki67 and blood vessel marker CD31 per high power field (p=0.0006 and 0.0008, respectively). Remarkably, tumor growth was completely absent after mammary fat pad implantation of R3230-GFP cells secreting the antagonist R103A-Epo protein (n=10 mice) compared to controls (183±46 mm3, n=8). Taken together, these data indicate that 1)-Epo is an important angiogenic factor that modulates tumor cell-induced angiogenesis and 2)-Suppression of tumor angiogenesis and progression by Epo blockade suggests that Epo may constitute a potential target for the therapeutic modulation of angiogenesis in cancer.

Characteristic Changes in Anti-erythropoietin Antibodies in a Peritoneal Dialysis Patient Who Developed Pure Red Cell Aplasia

Although cases of anti-erythropoietin (anti-EPO) antibody-induced pure red cell aplasia (PRCA) have been reported since 1998, the role of anti-EPO antibodies in the development of PRCA is controversial, with many conflicting opinions. Here, we present a patient who had serial serum anti-EPO antibody levels measured both before and after the development of PRCA to observe the relationship between serial changes in anti-EPO antibody and hemoglobin levels. Enzyme-linked immunosorbent assays were used for the detection of anti-EPO antibodies. The patient's serum anti-EPO antibody levels showed a characteristic change over time: negative before the development of PRCA, strongly positive when PRCA was diagnosed, decreasing after receiving prednisone, and eventually became negative. More importantly, an inversely close association between changes in serum anti-EPO antibody and hemoglobin levels was shown. The characteristic changes in this case indicate that anti-EPO antibody might have a role in the development of PRCA, and monitoring the titer of anti-EPO antibody may be of great value in the diagnosis and treatment of PRCA.

Efficient purification and preconcentration of erythropoietin in human urine by reusable immunoaffinity column

In this paper, an efficient method is proposed for purification and preconcentration of erythropoietin (EPO) in human urine samples. The EPO-specific immunoaffinity column (IAC) was generated by covalent immobilization of anti-EPO polyclonal antibodies on Sepharose 4B support. The EPO-binding capacity of the IAC was found to be about 2.0 microg (6.6IU) per 1.5 mL of gel and the activity recoveries of EPO at low concentrations of 7.8, 10 and 120 m IU/mL by the IAC were between 78 and 86%. Substantial cleanup effect was observed when the IAC was applied to human urine samples.

False-positive detection of recombinant human erythropoietin in urine following strenuous physical exercise

AbstractErythropoietin (Epo) is a glycoprotein hormone that promotes the production of red blood cells. Recombinant human Epo (rhEpo) is illicitly used to improve performance in endurance sports. Doping in sports is discouraged by the screening of athletes for rhEPO in urine. The adopted test is based on a combination of isoelectric focusing and double immunoblotting, and distinguishes between endogenous and recombinant human Epo. We show here that this widely used test can occasionally lead to the false-positive detection of rhEpo (epoetin-β) in postexercise, protein-rich urine, probably because the adopted monoclonal anti-Epo antibodies are not monospecific.

Detection of anti-EPO antibodies in human sera by a bridging ELISA is much more sensitive when coating biotinylated rhEPO to streptavidin rather than using direct coating of rhEPO

Sensitive and efficient methods for detecting anti-erythropoietin (anti-EPO) antibodies are needed for analysis and, above all, for large scale screening of human serum samples. ELISA is an attractive alternative to labor-intensive radioimmunoprecipitation assays but apparently conflicting reports question its sensitivity. We sought to resolve this issue by directly comparing different reported ELISA approaches to determine whether rhEPO-coating methods affect detection of anti-EPO antibodies. Investigators reporting low sensitivity had used ELISAs in which rhEPO was directly coated to microtiter plates while the high sensitivity ELISA used plate-bound streptavidin to bind biotinylated rhEPO. Using anti-EPO positive human sera, our results confirmed a large (100- to 300-fold) difference in sensitivity between the ELISAs and suggested that the inferiority of the low sensitivity ELISA was caused by the direct coating of rhEPO which may disrupt epitopes by masking recognition sites or introducing conformational changes. Thus, a bridging ELISA can be an appropriate and effective system for antibody analysis and screening of human sera with high sensitivity and specificity but only if performed with streptavidin binding of biotinylated antigen. This finding may also be more generally applicable to the detection of antibodies against other protein antigens.

Erythropoietin increases eNOS protein in the mouse heart.

It is generally accepted that induction of iNOS, but not eNOS, in the heart renders it less susceptible to the detrimental effects of an ischemia/reperfusion (I/R) challenge. However, there is some evidence to indicate that induction of eNOS in the heart (adenosine receptor agonist) can also provide protection against I/R-induced myocardial dysfunction and injury. We have recently shown that pretreatment of the heart with erythropoietin (EPO) can ameliorate the inflammation associated with an I/R challenge given 24 hrs later. The aim of the present study was to determine whether EPO can increase myocardial eNOS protein expression. C57BL/6 mice were treated with EPO (5,000 U/kg; intrperitoneally). Twenty four hrs later, the animals were euthanized, the hearts removed and prepared for Western blot analysis. Briefly, 5 μg of protein from heart tissue hydrolysates were resolved on 12.5% SDS-PAGE and transferred to polyvinylidiene fluoride membranes. After blocking with 5% non-fat milk, the membranes were blotted with an anti-EPO eNOS antibody. Our results indicate that EPO can induce eNOS protein expression in the myocardium. The increase in eNOS protein in the myocardium could explain the protective effects of EPO in I/R-induced myocardial dysfunction and injury. (MOP-13368; MGC-12816)