title: Development and evaluation of a new ELISA for the detection and quantification of antierythropoietin antibodies in human sera.
abstract: Assays for the analysis of antierythropoietin antibodies (anti-EPO Abs) currently suffer from a high degree of nonspecificity or are cumbersome and time consuming to perform. They are therefore not well suited for the analysis of large numbers of human sera samples, a task that has become increasingly important due to an increase in the number of patients developing anti-EPO Abs. The objective of this study was to develop and validate a sensitive and specific ELISA for the determination of anti-EPO Abs that would suit these purposes. In this new double antigen bridging ELISA, anti-EPO Abs bind via one site to recombinant human erythropoietin (rhEPO)-biotin immobilized to streptavidin-coated microtiter plates (MTPs) and by a second site to rhEPO labelled with digoxigenin (DIG). The amount of bound antibody is determined using an anti-DIG antibody coupled to peroxidase. A rabbit polyclonal anti-EPO Ab purified by immunoadsorption is used as reference antibody preparation. The dynamic range of this ELISA was 1-75 ng/ml per assay calibrated with the reference antibody preparation. The assay was specific for anti-EPO Abs and did not react with other immunoglobulins (Ig) present in human serum. The lower limit of detection (LLD) of the assay was 0.5 ng/ml, and the lower limit of quantitation (LLQ) was 1.0 ng/ml. Anti-EPO Abs could be detected in the sera of pure red cell aplasia (PRCA) patients. In contrast to previous reports, no anti-EPO Abs could be detected in the sera of patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjogren's syndrome (SS), or in the sera of dialysis patients.
authors.affiliation: Roche Diagnostics GmbH, Nonnenwaldstr. 2, 82372 Penzberg, Germany. wolfgang.hoesel@roche.com
authors.forename: W
authors.initials: W
authors.lastname: Hoesel
authors.forename: J
authors.initials: J
authors.lastname: Gross
authors.forename: R
authors.initials: R
authors.lastname: Moller
authors.forename: B
authors.initials: B
authors.lastname: Kanne
authors.forename: A
authors.initials: A
authors.lastname: Wessner
authors.forename: G
authors.initials: G
authors.lastname: Müller
authors.forename: A
authors.initials: A
authors.lastname: Müller
authors.forename: E
authors.initials: E
authors.lastname: Gromnica-Ihle
authors.forename: M
authors.initials: M
authors.lastname: Fromme
authors.forename: S
authors.initials: S
authors.lastname: Bischoff
authors.forename: A
authors.initials: A
authors.lastname: Haselbeck
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood* / Q000276:immunology; D001710:Biotin / Q000737:chemistry; D004076:Digoxigenin / Q000737:chemistry / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004921:Erythropoietin / Q000737:chemistry* / Q000276:immunology* / Q000627:therapeutic use; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000097:blood / Q000188:drug therapy; D009195:Peroxidase / Q000737:chemistry; D011817:Rabbits; D011994:Recombinant Proteins; D012010:Red-Cell Aplasia, Pure / Q000097:blood / Q000188:drug therapy; D012015:Reference Standards; D012016:Reference Values; D006435:Renal Dialysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012859:Sjogren's Syndrome / Q000097:blood / Q000188:drug therapy; D019809:Streptavidin / Q000737:chemistry
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; D004921:Erythropoietin; D001710:Biotin; D019809:Streptavidin; D009195:Peroxidase; D004076:Digoxigenin