Antibody-based protein multiplex platforms: Technical and operational challenges

Allison A. Ellington, Iftikhar J. Kullo, Kent R. Bailey, George G. Klee

Research output: Contribution to journalShort surveypeer-review

214 Scopus citations


BACKGROUND: The measurement of multiple protein biomarkers may refine risk stratification in clinical settings. This concept has stimulated development of multiplexed immunoassay platforms that provide multiple, parallel protein measurements on the same specimen. CONTENT: We provide an overview of antibody-based multiplexed immunoassay platforms and discuss technical and operational challenges. Multiplexed immunoassays use traditional immunoassay principles in which high-affinity capture ligands are immobilized in parallel arrays in either planar format or on microspheres in suspension. Development of multiplexed immunoassays requires rigorous validation of assay configuration and analytical performance to minimize assay imprecision and inaccuracy. Challenges associated with multiplex configuration include selection and immobilization of capture ligands, calibration, interference between antibodies and proteins and assay diluents, and compatibility of assay limits of quantification. We discuss potential solutions to these challenges. Criteria for assessing analytical multiplex assay performance include the range of linearity, analytical specificity, recovery, and comparison to a quality reference method. Quality control materials are not well developed for multiplexed protein immunoassays, and algorithms for interpreting multiplex quality control data are needed. SUMMARY: Technical and operational challenges have hindered implementation of multiplexed assays in clinical settings. Formal procedures that guide multiplex assay configuration, analytical validation, and quality control are needed before broad application of multiplexed arrays can occur in the in vitro diagnostic market.

Original languageEnglish (US)
Pages (from-to)186-193
Number of pages8
JournalClinical chemistry
Issue number2
StatePublished - Feb 1 2010

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical


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