Antibody Colocalization Microarray: A Scalable Technology for Multiplex Protein Analysis in Complex Samples

Abstract

DNA microarrays were rapidly scaled up from 256 to 6.5 million targets, and although antibody microarrays were proposed earlier, sensitive multiplex sandwich assays have only been scaled up to a few tens of targets. Cross-reactivity, arising because detection antibodies are mixed, is a known weakness of multiplex sandwich assays that is mitigated by lengthy optimization. Here, we introduce (1) vulnerability as a metric for assays. The vulnerability of multiplex sandwich assays to cross-reactivity increases quadratically with the number of targets, and together with experimental results, substantiates that scaling up of multiplex sandwich assays is unfeasible. We propose (2) a novel concept for multiplexing without mixing named antibody colocalization microarray (ACM). In ACMs, both capture and detection antibodies are physically colocalized by spotting to the same two-dimensional coordinate. Following spotting of the capture antibodies, the chip is removed from the arrayer, incubated with the sample, placed back onto the arrayer and then spotted with the detection antibodies. ACMs with up to 50 targets were produced, along with a binding curve for each protein. The ACM was validated by comparing it to ELISA and to a small-scale, conventional multiplex sandwich assay (MSA). Using ACMs, proteins in the serum of breast cancer patients and healthy controls were quantified, and six candidate biomarkers identified. Our results indicate that ACMs are sensitive, robust, and scalable.

Bulked sample analysis in genetics, genomics and crop improvement

“…Technically, microarray‐based high‐throughput analysis of protein–protein and other biomolecular interactions holds immense potential for multiplex interactome mapping and also great opportunity for an inclusive representation of the signal transduction pathways and networks (Gahoi et al ., ). Equipped with multiplexing, quantitative proteomics comes to the era of ‘ultra’ high‐throughput, making it possible to comprehensively compare all major tissues/organs of humans (Paulo et al ., ) and plants as well in a day.…”

“…Antibody colocalization microarray (ACM) as a novel concept for protein multiplexing without mixing has been used to quantify proteins in the serum of patients with breast cancer and healthy controls, with six candidate biomarkers identified (Pla-Roca et al, 2012). ACM involves a physical colocalization of both capture and detection antibodies, spotting of the capture antibodies, and sample incubation, followed by spotting of the detection antibodies.…”

Cancer proteomics: developments in technology, clinical use and commercialization

“…The reason for their unpopularity is that the sandwich antibody protocol makes these assays vulnerable to cross-reactivity, which increases quadratically with the number of targets, thus limiting the proteins that can be analyzed to tens of proteins. To remedy this, Pla-Roca et al tested a novel modification to the antibody co-localization microarray that reduces cross-reactivity by spotting capture and detection antibodies to the same two-dimensional coordinate [12]. If successful, this approach could deliver higher sensitivity without sacrificing throughput.…”

Imaging Flow Cytometry

“…Recently, new approaches, such as antibody colocalization microarrays and reverse protein assays, have been developed (Kornblau et al 2009;Brennan et al 2010;Pla-Roca et al 2012). The phosphorylation of a protein is the principal cellular mechanism regulating protein function and is controlled by protein kinases and phosphatases.…”