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Development of a Sensitive Bead-Based Assay for Enhanced Monoclonal Antibody Detection

Published online by Cambridge University Press:  01 July 2011

Manuel E. Ruidíaz ,
Natalie Mendez ,
Ana B. Sanchez ,
Bradley T. Messmer  and
Andrew C. Kummel
Manuel E. Ruidíaz
Affiliation:
Department of Bioengineering, University of California, San Diego, La Jolla (UCSD), CA 92093, U.S.A.
Natalie Mendez
Affiliation:
Division of Biological Sciences, UCSD, La Jolla, CA, 92093, U.S.A.
Ana B. Sanchez
Affiliation:
Moores Cancer Center, UCSD, La Jolla, CA 92093, U.S.A
Bradley T. Messmer
Affiliation:
Moores Cancer Center, UCSD, La Jolla, CA 92093, U.S.A
Andrew C. Kummel
Affiliation:
Department of Chemistry and Biochemistry, UCSD, La Jolla, CA 92093, U.S.A
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Abstract

Monoclonal antibodies are increasingly used in the treatment of cancer due to their enhanced targeting and immune system stimulation properties. Dosage guidelines typically do not account for personal cancer load or metabolism, thereby possibly affecting treatment outcome or causing unwanted side effects. The requirement for an assay that can quickly and precisely measure the concentration of the monoclonal antibody in a serum sample of a patient during therapy is unmet. A bead-based assay with peptide antigen mimetics has been developed to rapidly determine the concentration of antibody drug present in serum specimens with high sensitivity. Alemtuzumab (anti-CD52) and rituximab (anti-CD20) antigen mimetic peptides, as discovered by phage display, were synthesized on 10 um TentaGel resin beads using conventional solid phase peptide synthesis techniques. The beads were modified to allow for multiplexing and microfluidic handling via fluorescent labeling and magnetic functionalization. The antigen-displaying fluoromagnetic particles were incubated with spiked serum samples which allowed free antibody to be captured. Primary antibody detection was performed on alemtuzumab while rituximab detection was used to compensate for non-specific serum binding to the beads. After washing, the beads were incubated with a fluorescently tagged secondary label for detection by flow cytometry. (Results) A fast, low cost, specific assay has been developed with several key techniques which allows detection at low concentration (0.1ug/ml) of spiked samples. Primary to achieving this detection limit was the implementation of a compensation scheme where two antigen mimetic peptides behave linearly (R2=0.996) which enables the calculation of the zero response of the antigen mimetic peptide of interest (alemtuzumab antigen mimetic) while measuring the zero response of the compensatory antigen mimetic peptide (rituximab antigen mimetic) during primary assay measurement. This reduces fluorescence response variation due to variations present due to sample preparation, storage and different patients because of the equivalent interactions these effects have on the compensatory beads. The developed assay is therefore robust against serum variation and enables a lower limit of detection.

Keywords

biomedicalbiomimetic (chemical reaction)magnetic
Type
Research Article
Information
MRS Online Proceedings Library (OPL) , Volume 1346: Symposium AA – Micro- and Nanofluidic Systems for Materials Synthesis, Device Assembly and Bioanalysis , 2011 , mrss11-1346-aa15-07
Copyright
Copyright © Materials Research Society 2011

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