title: Measurement of anti-erythropoiesis-stimulating agent IgG4 antibody as an indicator of antibody-mediated pure red cell aplasia.
abstract: Patients treated with erythropoietin-based erythropoiesis-stimulating agents (ESAs) can develop a rare but life-threatening condition called antibody-mediated pure red cell aplasia (amPRCA). The antibody characteristics in a nephrology patient with amPRCA include high antibody concentrations with neutralizing activity and a mixed IgG subclass including anti-ESA IgG4 antibodies. In contrast, anti-ESA IgG4 antibody is generally not detected in baseline samples and antibody-positive non-PRCA patients. Therefore, we validated a highly sensitive immunoassay on the ImmunoCAP 100 instrument to quantitate anti-ESA IgG4 antibodies using a human recombinant anti-epoetin alfa (EPO) IgG4 antibody as a calibrator. The biotinylated ESA was applied to a streptavidin ImmunoCAP, and bound anti-ESA IgG4 antibodies were detected using a β-galactosidase-conjugated mouse anti-human IgG4 antibody. The validated assay was used to detect anti-ESA IgG4 in amPRCA and non-PRCA patients. The immunoassay detected 15 ng/ml of human anti-EPO IgG4 antibody in the presence of a 200 M excess of human anti-ESA IgG1, IgG2, or IgM antibody and tolerated 2 μg/ml of soluble erythropoietin. All patient samples with confirmed amPRCA had measurable anti-ESA IgG4 antibodies. In addition, 94% (17/18) of non-PRCA patient samples were antibody negative or had below 15 ng/ml of anti-ESA IgG4 antibodies. This novel immunoassay can measure low-nanogram quantities of human anti-ESA IgG4 antibodies in the presence of other anti-ESA antibodies. An increased concentration of anti-ESA IgG4 antibody is associated with the development of amPRCA. We propose that the measurement of anti-ESA specific IgG4 antibodies may facilitate early detection of amPRCA in patients receiving all ESAs structurally related to human erythropoietin.
authors.affiliation: Department of Clinical Immunology-Medical Sciences, Amgen, Thousand Oaks, CA, USA.
authors.forename: Dohan K
authors.initials: DK
authors.lastname: Weeraratne
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Kuck
authors.forename: Narendra
authors.initials: N
authors.lastname: Chirmule
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Mytych
mesh_terms: D057134:Antibodies, Neutralizing / Q000097:blood*; D001323:Autoantibodies / Q000097:blood*; D019411:Clinical Laboratory Techniques / Q000379:methods*; D004920:Erythropoiesis / Q000187:drug effects / Q000276:immunology; D004921:Erythropoietin / Q000276:immunology*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007074:Immunoglobulin G / Q000097:blood*; D012010:Red-Cell Aplasia, Pure / Q000175:diagnosis*; D012680:Sensitivity and Specificity
chemical_list: D057134:Antibodies, Neutralizing; D001323:Autoantibodies; D007074:Immunoglobulin G; D004921:Erythropoietin