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  • Review Article
  • Published:

Multiplexed protein measurement: technologies and applications of protein and antibody arrays

Key Points

  • Multiplexed protein measurement is a rapidly advancing field that has the broadest potential to transform drug discovery and development in the next ten years of any existing 'omics' technology.

  • Multiplexed protein measurement is logical for biological discovery with proteins because they function within networks, pathways, complexes and families. The activity of an individual protein is dependent not only on its abundance, but also on the effects of interacting proteins, modifying proteins, and antagonistic and synergistic proteins.

  • However, until recently, specific, sensitive, multiplexed protein measurement has not been generally performed because of technical challenges, including the diverse physicochemical properties of proteins, their lability and interferences introduced as a by-product of multiplexed measurement reagents.

  • Nevertheless, reports of discoveries made using multiplexed protein measurment technologies are starting to drive adoption of these approaches, and this article reviews these technologies, emerging standards, applications and remaining challenges.

  • Four main applications that follow a logical research and development succession are discussed: surveys of changes in protein abundance; modelling networks, pathways, physiological and disease states; biomarker validation; and clinical diagnostics.

  • The two principal types of technologies in use today in these applications are mass spectrometry and protein arrays, both of which discussed here, with a focus on protein arrays.

  • Three broad categories of antibody microarray experimental formats are described: direct labelling, single-capture antibody experiments; dual antibody, sandwich immunoassays; and antigen or peptide capture arrays, with single read-out antibodies. Advantages and disadvantages of these formats are compared.

  • The process of developing biomarkers based on multiplexed protein measurement into a clinical diagnostic test is discussed. The first step is multiplexed immunoassay development, in which optimized, multiplexed assays are developed for validated biomarkers and transferred to an immunodiagnostic platform. The second step is in vitro diagnostic development, driven by regulatory, manufacturing and marketing considerations.

Abstract

The ability to measure the abundance of many proteins precisely and simultaneously in experimental samples is an important, recent advance for static and dynamic, as well as descriptive and predictive, biological research. The value of multiplexed protein measurement is being established in applications such as comprehensive proteomic surveys, studies of protein networks and pathways, validation of genomic discoveries and clinical biomarker development. As standards do not yet exist that bridge all of these applications, the current recommended best practice for validation of results is to approach study design in an iterative process and to integrate data from several measurement technologies. This review describes current and emerging multiplexed protein measurement technologies and their applications, and discusses the remaining challenges in this field.

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Figure 1: Schematic representation of the two antibody microarray experimental formats.
Figure 2: Schematic representation of antigen or peptide capture arrays.
Figure 3: Steps for the development of validated biomarkers into a clinical diagnostic test.

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Acknowledgements

A Deo lumen, ab amicis auxiliam (to God for illumination, to friends for help). This work was partially supported by a National Institutes of Health grant. Thanks to J. Huntley, D. Gessler and the referees for critically reviewing this manuscript.

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FURTHER INFORMATION

PRoteomics IDEntifications (PRIDE)

Expert Protein Analysis System

Plasma Proteome Database

Global Proteome Machine

Human Protein Reference Database

PhysioNet

Glossary

Longitudinal study

A study in which measurements are made over a time course within an individual or individuals.

Cross-sectional study

A study in which measurements are made at a single time point in many individuals.

Single nucleotide polymorphisms

A specific location in a DNA sequence at which different people can have a different DNA base. Differences in a single base could change the protein sequence, leading to disease, or have no known consequences.

Cytokine

Cytokines comprise several hundred small, soluble proteins that are powerful mediators of target cell activities such as migration, activation, phagocytosis, proliferation and apoptosis. Cytokines are secreted by producer cells into extracellular fluids, and act on target cells through binding to specific cell-surface receptors.

Positive predictive value

(PPV). The probability that the patient has the disease when restricted to those who test positive.

Negative predictive value

(NPV). An assessment of reliability of a negative test. The probability that the patient does not have the disease when restricted to those who test negative.

21 CFR compliant

In compliance with Title 21 of the Code of Federal Regulations. This is the principal law enforced by the US FDA.

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Kingsmore, S. Multiplexed protein measurement: technologies and applications of protein and antibody arrays. Nat Rev Drug Discov 5, 310–321 (2006). https://doi.org/10.1038/nrd2006

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