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Clinical and Vaccine Immunology, September 2007, p. 1165-1172, Vol. 14, No. 9
1071-412X/07/$08.00+0     doi:10.1128/CVI.00157-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Feasibility of a Multiplex Flow Cytometric Bead Immunoassay for Detection of Anti-Epoetin Alfa Antibodies{triangledown}

John Ferbas,1* John Thomas,1 John Hodgson,2 Amitabh Gaur,2 Nicole Casadevall,3 and Steven J. Swanson1

Amgen, Inc., Thousand Oaks, California,1 BD Biosciences, San Diego, California,2 Hôpital de l'Hôtel-Dieu, Service d'Hématologie Biologique, Paris, France3

Received 11 April 2007/ Returned for modification 18 May 2007/ Accepted 5 July 2007

Immunogenicity profiles of recombinant therapeutic proteins are important to understand because antibodies raised against these molecules may have important clinical sequelae. The purpose of the present study was to demonstrate that a flow cytometric bead array could be used to detect clinically relevant antibodies with specificity to such therapeutics. We chose to evaluate well-characterized specimens from persons treated with epoetin alfa that developed antibody-mediated pure red blood cell aplasia as a means to demonstrate the utility of this platform. Our data show that this assay is capable of detecting anti-epoetin alfa antibodies with a relative antibody concentration of 50 ng/ml, where 25 of 25 sera spiked with antibodies at this concentration scored positive. Moreover, the assay was designed to include positive and negative control beads for each specimen that is processed to ensure the specificity of the signal when detected. Measurement of interassay precision supports quantitative estimates of relative antibody concentrations in the range of 313 to 5,000 ng/ml, where the percent coefficient of variation did not exceed 20%. With respect to clinical specimens, antibodies with specificity for epoetin alfa could be easily detected in a set of specimens from persons with pure red blood cell aplasia that had prior exposure to the EPREX brand of recombinant epoetin alfa. Further development and validation of this approach may facilitate successful widespread application of the method for detection of anti-epoetin alfa antibodies, as well as antibodies directed against other recombinant therapeutic proteins.

* Corresponding author. Mailing address: Department of Clinical Immunology, Amgen Inc., One Amgen Center Drive, Mailstop 30E-3-C, Thousand Oaks, CA 91320-1799. Phone: (805) 447-2867. Fax: (805) 480-1306. E-mail: jferbas{at}amgen.com

{triangledown} Published ahead of print on 18 July 2007.

Clinical and Vaccine Immunology, September 2007, p. 1165-1172, Vol. 14, No. 9
1071-412X/07/$08.00+0     doi:10.1128/CVI.00157-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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