Detection of neutralizing antibodies to erythropoietin by inhibition of rHuEPO-stimulated EGR1 gene expression in the UT-7/EPO cell line

J Immunol Methods. 2013 Jan 31;387(1-2):191-8. doi: 10.1016/j.jim.2012.10.014. Epub 2012 Nov 8.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Neutralizing / analysis*
  • Antibodies, Neutralizing / immunology
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Dose-Response Relationship, Drug
  • Early Growth Response Protein 1 / genetics*
  • Erythropoietin / genetics
  • Erythropoietin / immunology
  • Erythropoietin / pharmacology*
  • Gene Expression / drug effects*
  • Gene Expression / immunology
  • Humans
  • Immune Sera / immunology
  • Immune Sera / pharmacology
  • Immunologic Techniques / methods
  • Leukemia, Erythroblastic, Acute / genetics
  • Leukemia, Erythroblastic, Acute / immunology
  • Leukemia, Erythroblastic, Acute / pathology
  • Recombinant Proteins / pharmacology*
  • Red-Cell Aplasia, Pure / blood
  • Red-Cell Aplasia, Pure / immunology
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction
  • Serum / immunology
  • Sheep
  • Time Factors


  • Antibodies, Neutralizing
  • EGR1 protein, human
  • Early Growth Response Protein 1
  • Immune Sera
  • Recombinant Proteins
  • Erythropoietin