Antibody Colocalization Microarray: A Scalable Technology for Multiplex Protein Analysis in Complex Samples
2011; Elsevier BV; Volume: 11; Issue: 4 Linguagem: Inglês
10.1074/mcp.m111.011460
ISSN1535-9484
AutoresMateu Pla‐Roca, Rym Feriel Leulmi, Saule Tourekhanova, Sébastien Bergeron, Véronique Laforte, Emmanuel Moreau, Sara J.C. Gosline, Nicholas Bertos, Michael Hallett, M. Park, David Juncker,
Tópico(s)Gene expression and cancer classification
ResumoDNA 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. 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. Semiconductors are the paradigm for scalable technologies. These grew exponentially over four decades by doubling the density of elements and processing speed roughly every two years following Moore's law (1Moore G.E. No exponential is forever: but "Forever" can be delayed!.Proceedings of the IEEE Solid-State Circuits Conference (ISSCC). 2003; vol. 21: 20-23Google Scholar). DNA microarrays, introduced in the early nineties, were also scalable and expanded from initially 256 (2Pease A.C. Solas D. Sullivan E.J. Cronin M.T. Holmes C.P. Fodor S.P. Light-generated oligonucleotide arrays for rapid DNA sequence analysis.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5022-5026Crossref PubMed Scopus (1193) Google Scholar) to 6.5 million probes per chip (3Ragoussis J. Elvidge G. 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Biotechnol. 2011; 29: 625-634Crossref PubMed Scopus (295) Google Scholar). These results validate MS for identifying biomarkers across the human proteome, but both pipelines required (i) using multiple MS instruments and advanced protocols, (ii) weeks and months of instrument time for data acquisition, (iii) depletion of abundant proteins in the plasma, which is labor intensive and may result in loss of biomarker proteins, and (iv) high concentrations of the candidate biomarkers in the sample for the first step of the validation pipeline, which may not be practical for some diseases. Mass spectrometry limit of detection multiplex sandwich assay antibody colocalization microarray antibody capture antibody detection antibody streptavidin phosphate buffered saline. The sandwich immunoassay (Fig. 1A), which is widely used for enzyme-linked immunosorbent assays (ELISAs), is the "gold standard" for detecting proteins at low concentrations. Sandwich immunoassays, in contrast to direct labeling assays, are tolerant to cross-reactivity, which does not necessarily lead to false positive signals or noise. The dual binding of capture (cAb) and detection antibody (dAb) to two different epitopes of the same protein prevents a single cross-reaction, or the nonspecific binding of a protein to a spot, from generating a false positive signal because the dAb will not bind. Sandwich assays have been continuously improved and single molecules can now be detected (19Rissin D.M. Kan C.W. Campbell T.G. Howes S.C. Fournier D.R. Song L. Piech T. Patel P.P. Chang L. Rivnak A.J. Ferrell E.P. Randall J.D. Provuncher G.K. Walt D.R. Duffy D.C. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations.Nat. Biotechnol. 2010; 28: 595-599Crossref PubMed Scopus (1330) Google Scholar), but in samples with complex matrices such as blood, "interference" still occurs and the LOD in clinical tests is limited to high femtomolar ( 50% signal variation was observed among replicate spots were discarded. The Wilcoxon rank sum test and subsequence box-plots were produced by R/Bioconductor statistical software (http://www.bioconductor.org). Ward's clustering algorithm and the Euclidean distance metric were used for hierarchical clustering. All values below half of the calculated LOD were adjusted to ½ LOD for clustering and t test analysis. As discussed above, sandwich assays are characterized by their tolerance to some cross-reactivity among cAb and unrelated proteins because even in the event of cross-reactivity, no detectable signal is produced because the dAb does not bind to the protein. We introduce the concept of "liability pairs," which designates the combination of Ab-Ab, Ab-protein, or protein-protein in which a single cross-reactive binding or interaction among proteins will translate into a false positive signal (or background noise). Liability pairs do not exist in single-plex sandwich assays, but arise in MSAs following the application of a mixture of dAbs to the array. Each dAb will interact with all immobilized molecules on any of the spots. Thus, cross-reactive binding of any analyte to a cAb, protein-protein interactions among analytes, or direct cross-reactive binding of any dAb to any of the proteins immobilized on a spot will generate a false positive signal. The number of liability pairs for an array with N targets can be computed by combinatorial enumeration of each pair according to the scenarios illustrated in Fig. 2: (1) dAb-antigen, (2) dAb-cAb and (3) dAb-dAb, (4) cAb-antigen and (5) antig
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