title: Detection of neutralizing antibodies to erythropoietin by inhibition of rHuEPO-stimulated EGR1 gene expression in the UT-7/EPO cell line.
abstract: Recombinant erythropoietin (rHuEPO) is used extensively to treat anaemia associated with chronic kidney disease. However, the development of neutralizing antibodies (NAbs) to rHuEPO can result in the development of antibody-mediated pure red cell aplasia (PRCA). The detection of NAb in patient sera by in vitro bioassay relies on the inhibition of a cellular response to rHuEPO. Current bioassays for rHuEPO measure proliferation in responsive cell lines such as the erythroleukaemic cell lines, UT-7 and UT-7/EPO, the latter sensitized to EPO. Using these cell lines, we show the dose-responsive induction of both PIM1 and EGR1 gene expression in UT-7 cells and of EGR1 in UT-7/EPO cells. The expression of EGR1 in UT-7/EPO cells in response to rHuEPO was comparable to the proliferative response measured by (3)H-thymidine incorporation and could be inhibited by serum from a patient with NAb-mediated PRCA in a dilution-dependent manner. Bioassays based on the induction of endogenous gene expression are comparable to current bioassays but are considerably quicker given that incubation time is decreased from 2-3 days to 50 min. Measurement of EGR1 gene expression in response to rHuEPO in UT-7/EPO cells offers a rapid, non-radioactive and automatable alternative to current assays for the detection of rHuEPO NAbs.
authors.affiliation: Biotherapeutics Group, National Institute of Biological Standards and Control, South Mimms, Potters Bar, Herts EN6 3QG, UK. Jackie.Ferguson@nibsc.hpa.org.uk
authors.forename: Jackie
authors.initials: J
authors.lastname: Ferguson
authors.forename: Chris
authors.initials: C
authors.lastname: Bird
authors.forename: Meenu
authors.initials: M
authors.lastname: Wadhwa
authors.forename: Chris
authors.initials: C
authors.lastname: Burns
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000032:analysis* / Q000276:immunology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D051766:Early Growth Response Protein 1 / Q000235:genetics*; D004921:Erythropoietin / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects* / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000276:immunology / Q000494:pharmacology; D007158:Immunologic Techniques / Q000379:methods; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics / Q000276:immunology / Q000473:pathology; D011994:Recombinant Proteins / Q000494:pharmacology*; D012010:Red-Cell Aplasia, Pure / Q000097:blood / Q000276:immunology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044967:Serum / Q000276:immunology; D012756:Sheep; D013997:Time Factors
chemical_list: D057134:Antibodies, Neutralizing; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D007106:Immune Sera; D011994:Recombinant Proteins; D004921:Erythropoietin