title: The development and validation of a sensitive, dual-flow cell, SPR-based biosensor immunoassay for the detection, semi-quantitation, and characterization of antibodies to darbepoetin alfa and epoetin alfa in human serum.
abstract: A surface plasmon resonance (SPR)-based biosensor immunoassay was developed and validated using the Biacore 3000 instrument to detect, semi-quantitate, and characterize serum antibodies against darbepoetin alfa (Aranesp) and epoetin alfa (EPOGEN). In this sensitive, dual-flow cell assay, epoetin alfa and darbepoetin alfa are covalently immobilized onto consecutive flow cells of a carboxymethyl dextran-coated sensor chip. Diluted human serum samples are injected sequentially over both surfaces. The binding of serum antibodies to the immobilized proteins are detected and recorded in real time based on the principles of SPR. Furthermore, antibody binding is confirmed with a secondary anti-human immunoglobulin antibody. Positive samples are further characterized to determine the relative concentration of the antibodies using an affinity-purified, rabbit anti-epoetin alfa antibody as a reference control. The assay can detect 80ng/ml and 100ng/ml of antibody to epoetin alfa and darbepoetin alfa, respectively. The dynamic range of the assay is from 0.078microg/ml to 10microg/ml using a rabbit antibody with demonstrated accuracy and intra- and inter-assay precision. Approximately 80 serum samples can be analyzed on each sensor chip while maintaining a stable baseline and consistent immunological reactivity. The analysis of serum samples from subjects administered with epoetin alfa or darbepoetin alfa provided evidence that the assay can detect varying concentrations of antibodies of different off rates, isotypes, and IgG subclasses.
authors.affiliation: Department of Clinical Immunology, Medical Sciences, Amgen Inc., Thousand Oaks, CA 91320, USA. dmytych@amgen.com
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Mytych
authors.forename: Stephon
authors.initials: S
authors.lastname: La
authors.forename: Troy
authors.initials: T
authors.lastname: Barger
authors.forename: John
authors.initials: J
authors.lastname: Ferbas
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Swanson
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood* / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods; D000068256:Darbepoetin alfa; D004355:Drug Stability; D004356:Drug Storage; D000068817:Epoetin Alfa; D004921:Erythropoietin / Q000031:analogs & derivatives* / Q000097:blood*; D005615:Freezing; D006397:Hematinics / Q000097:blood*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*
chemical_list: D000906:Antibodies; D006397:Hematinics; D011994:Recombinant Proteins; D004921:Erythropoietin; D000068256:Darbepoetin alfa; D000068817:Epoetin Alfa