The detection of anti-erythropoietin antibodies in human serum and plasma. Part I. Validation of the protocol for a radioimmunoprecipitation assay

Tacey, R.; Greway, A.; Smiell, J.; Power, D.; Kromminga, A.; Daha, M.; Casadevall, N.; Kelley, M.

Journal of Immunological Methods 283(1-2): 317-329


ISSN/ISBN: 0022-1759
PMID: 14659922
DOI: 10.1016/j.jim.2003.09.003
Accession: 012642218

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Rare cases of unexplained sudden severe anemia or red cell aplasia and resistance to recombinant human erythropoietin (rHuEPO) in patients with chronic renal failure (CRF) have been attributed to the development of anti-EPO antibodies. The development and validation of a radioimmunoprecipitation (RIP) assay to detect human anti-EPO antibodies in serum or plasma has been hampered by the lack of purified antibody to fully characterize and validate the assay. We have prepared an affinity-purified human antibody to EPO and used the antibody to characterize and validate a sensitive and reproducible RIP assay that can qualitatively measure anti-EPO antibody in serum or plasma samples. The lower limit of detection of the assay is 8 ng/ml of purified antibody. The threshold for detecting antibody is > or =0.9% cpm bound. The precision of the assay using purified antibody standards ranges from 5.8% to 15.3% and the precision of the assay using dilutions of the positive control ranges from 15.9% to 18.7%. EPO in the samples did not interfere with detection of the anti-EPO antibody except at high concentrations.