Development and evaluation of a new ELISA for the detection and quantification of antierythropoietin antibodies in human sera

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. (C) 2004 Elsevier B.V. All rights reserved.