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

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.

Figure 1

Schematic representation of the two antibody microarray experimental formats. a | Direct labelling, single-capture antibody experiments. All proteins in a sample are labelled (red haloes), thereby providing a means for detecting bound proteins following incubation on an antibody microarray. b | Dual-antibody (capture and read-out antibody) sandwich immunoassays. Proteins captured on an antibody microarray are detected by a cocktail of tagged detection antibodies, which are matched to the spotted antibodies. The detector antibody tag is then measured by binding of a labelled (red halo) read-out antibody.

Figure 2

Schematic representation of antigen or peptide capture arrays. Antigens are printed on the array, which is then incubated with an experimental sample containing antibodies. Binding of the antibodies to the antigens can then be detected by binding a secondary, anti-immunoglobulin (a read-out antibody) that has been fluorescently labelled (red halo). Ig, immunoglobulin.

Figure 3

Steps for the development of validated biomarkers into a clinical diagnostic test. Optimized, multiplexed assays are developed for validated biomarkers and transferred to an immunodiagnostic platform. Then specifications are determined regarding analytical and clinical performance. Finally, in vitro diagnostic development is driven by regulatory, manufacturing and marketing considerations. CLIA, Clinical Laboratory Improvement Amendment (the US law to ensure quality laboratory testing); GMP, good manufacturing practice.