Validation of the BIACORE 3000 platform for detection of antibodies against erythropoietic agents in human serum samples

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Abstract
SUMMARYObjective: To develop a validated BIACORE immunoassay for the detection and characterization of serum antibodies with specificity for erythropoietic molecules (e.g. darbepoetin alfa).Methods: New Zealand White rabbits (n = 8) were immunized by an intramuscular injection of darbepoetin alfa/adjuvant at 0,4,6, and 8 weeks. Serum was collected for 6 weeks after final injection and pooled for affinity purification. Antibody immunoassay measurements were performed using a BIACORE 3000 with darbepoetin alfa immobilized to the biosensor surface. Human serum samples were spiked with the affinity-purified rabbit antibody to develop and validate the BIACORE immunoassay.Results: The assay was shown to be stable through 180 sample/regeneration cycles and had a threshold of 45.8 response units. The validated limit of detection was 0.40|ig/ml in 100% human serum. The method was robust, with variability notexceeding a 20% coefficient of variation, well within acceptable limits for typical immunoassays.Conclusion:...