title: Strategy to confirm the presence of anti-erythropoietin neutralizing antibodies in human serum.
abstract: Functional cell-based assays are the preferred method to test for the presence of anti-rHuEPO neutralizing antibodies (NAbs). However, due to the unpredictable nature of test serum matrix effects on cell-based assays, confirmatory assays are essential for verifying NAb positive results observed during the course of sample testing. The cell-based assay used for the detection of NAbs described by Wei et al. [1] used 32D-EPOR cells, a murine myeloid cell line transfected with the human EPO receptor (EPOR). The 32D-EPOR cell line responded to either rHuEPO or murine interleukin 3 (mIL-3) with proliferation. NAbs were expected to only inhibit rHuEPO-induced cell proliferation and not mIL-3 induced proliferation. Due to reliance on proliferation, the results from this cell-based assay can be confounded by the presence of non-antibody inhibitory serum factors. This paper describes a strategy for confirming that the inhibition of rHuEPO-induced proliferation in a cell-based assay is only attributable to NAbs. The strategy of antibody depletion uses a resin mixture composed of Protein G Sepharose and Protein L Sepharose (Protein G/L resin) to significantly reduce the concentration of immunoglobulins of IgG, IgM and IgA isotypes from human serum prior to testing in the cell-based assay. If the reduction in immunoglobulins in a serum sample corresponds to a reduction in inhibition of EPO-induced proliferation, it would infer that EPO neutralizing activity is antibody-mediated and not due to non-antibody inhibitory serum factors.
authors.affiliation: Department of Clinical Immunology, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
authors.forename: Sergio
authors.initials: S
authors.lastname: Sanchez
authors.forename: Troy
authors.initials: T
authors.lastname: Barger
authors.forename: Lei
authors.initials: L
authors.lastname: Zhou
authors.forename: Michael
authors.initials: M
authors.lastname: Hale
authors.forename: Daniel
authors.initials: D
authors.lastname: Mytych
authors.forename: Shalini
authors.initials: S
authors.lastname: Gupta
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Swanson
authors.forename: Francesca
authors.initials: F
authors.lastname: Civoli
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D057134:Antibodies, Neutralizing / Q000737:chemistry*; D015374:Biosensing Techniques; D002460:Cell Line; D049109:Cell Proliferation; D004921:Erythropoietin / Q000737:chemistry*; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007377:Interleukin-3 / Q000378:metabolism; D008297:Male; D051379:Mice; D017467:Receptors, Erythropoietin / Q000737:chemistry; D012010:Red-Cell Aplasia, Pure / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry; D044967:Serum / Q000378:metabolism*
chemical_list: D000906:Antibodies; D057134:Antibodies, Neutralizing; D007377:Interleukin-3; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D012685:Sepharose