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Multiplexed Protein Detection by Proximity Ligation for Cancer

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Multiplexed Protein Detection by Proximity Ligation for Cancer BRIEF COMMUNICATIONS polyclonal antibody per target protein, and we apply the assay in Multiplexed protein clinical blood samples for biomarker validation. The technical ability to analyze nucleic acids is far more detection by proximity advanced than that for proteins. Proximity ligation has been developed in an attempt to bridge this gap and thereby improve methods ligation for cancer the limit of detection, specificity, dynamic range and multiplex capability5,6. The technology is a unique approach for protein biomarker validation quantification (often seen in nucleic acid detection) wherein the analyte promotes the creation of a new and distinct reporter .com/nature e 1 1 1 molecule. This is in contrast to heterogenous methods, which Simon Fredriksson , William Dixon , Hanlee Ji , require washes to remove unbound secondary reporters after 2 1 .natur Albert C Koong , Michael Mindrinos & solid-phase capture. The homogenous, or liquid phase version of w Ronald W Davis1 the technology uses a pair of proximity probes each composed of an antibody linked to an oligonucleotide. As the two antibodies bind We present a proximity ligation–based multiplexed protein the protein analyte in solution, the local concentration of the two http://ww detection procedure in which several selected proteins can be corresponding oligonucleotides increases. This allows the hybridi- detected via unique nucleic-acid identifiers and subsequently zation of one connecting oligonucleotide to both probes, thus oup r quantified by real-time PCR. The assay requires a 1-llsample, enabling an enzymatic ligation joining the 3¢ end of the first G has low-femtomolar sensitivity as well as five-log linear range probe with the 5¢-end of the second probe. This leads to the and allows for modular multiplexing without cross-reactivity. formation of a unique target reporter amplicon containing specific lishing The procedure can use a single polyclonal antibody batch for molecular bar codes, a frequently used procedure for multiplexed b each target protein, simplifying affinity-reagent creation for detection of nucleic acids7. These molecular bar codes serve as Pu new biomarker candidates. primer sites, of which some are universal for all protein analytes, whereas others are target-specific for quantification by real-time Protein-based biomarkers in blood hold a great promise as PCR. The assay reporter signal is dependent on a proximal and dual Nature 7 diagnostic markers indicative of disease states and outcomes in recognition of each target analyte providing high specificity. We clinical cancer management. To validate large sets of candidate outline the multiplex detection procedure in Figure 1, and detailed 200 © markers in bio-banked sample collections, multiplexed and sensi- protocols are available in the Supplementary Methods online. tive detection technologies with low sample consumption are To suppress the level of background ligations, we incubated the required. Such technologies will not only be critical for the proximity probes at subsaturating concentrations during the initial discovery of biomarker panels potentially leading to increased incubation with the sample. Therefore, the sensitivity and dynamic predictive power in diagnostics but may eventually permit early range of the assay is dependent on the affinity of the antibody6. stage disease detection1,2. Most conventional immunoassays rely on a solid support for capture of the target protein and for the removal of excess Sample Bar-coded amplicons Protein abundance 10 secondary reporter antibody by washing. The sensitivity of these VEGF 1. …ATATGCATCG… IL-4 …GATCGATGCG… qPCR 1 sandwich assays is limited by the nonspecific binding of the IL-10 Proximity ligation …GTTTGAGGCG… α IL-1 …GTACGGCATC… 0.1 secondary reporter to the surface of the solid support. Also, any TNFα 2. …TAGATCCGCG… single noncognate binding event by a detection antibody will give IL-7 …TGATCCGTAG… 0.01 IL-1 IL-4 IL-10 TNF IL-7 rise to a false positive signal. This is especially challenging when VEGF performing multiplexed reactions with many detection antibodies, requiring extensive and careful optimization through selection of Figure 1 | Schematic outline of multiplexed proximity ligation. The sample particular antibody combinations to minimize nonspecific cross- is first incubated with sets of proximity probes, antibodies equipped with oligonucleotides in step 1. A connector oligonucleotide is then added in reactivity3,4. To reduce this scalability bottleneck and lower sample step 2 uniting proximal sequences forming unique bar-coded amplicons consumption, we configured a protein detection technology, the representing each target protein. This is followed by preamplification of all proximity ligation assay, to remove potential cross-reactive signals ligation products. Finally, the surrogate markers for the proteins comprised when performed in multiplex. In multiplex, we also show the assay within the pool are analyzed by real-time PCR in a miniaturized format, can operate with simple affinity reagents such as one single batch of generating quantitative protein-abundance data. 1Stanford Genome Technology Center, Bio-X, 318 Campus Dr., Stanford, California 94305, USA. 2Department of Radiation Oncology, Stanford, California 94305, USA. Correspondence should be addressed to S.F. ([email protected]) and R.W.D. ([email protected]). RECEIVED 20 OCTOBER 2006; ACCEPTED 23 JANUARY 2007; PUBLISHED ONLINE 18 MARCH 2007; DOI:10.1038/NMETH1020 NATURE METHODS | VOL.4 NO.4 | APRIL 2007 | 327 BRIEF COMMUNICATIONS quantitative PCR (qPCR) detection. In the presence of a complex a 108 VEGF mixture containing chicken plasma, we spiked 10 pM of one of the IL-4 7 10 IL-10 six target proteins into the incubation together with all the six IL-1α 6 α probe pairs and recorded the signal over background for each 10 TNF IL-7 reaction (Fig. 2b). We observed no antibody cross-reactivity even 105 without optimization of conditions or requirement of selection of particular antibody combinations. This demonstrates potential for 4 10 scalability beyond that of solid phase–based arrays, presently qPCR amplicons B 103 limited to 10–20-plex. In comparison, multiplexing with multi- color bead–based assays has in some cases been reported to result in (estimated number of molecules) (estimated number 102 a loss of sensitivity, resulting in certain proteins becoming unde- tectable in plasma9. As cross-reactive events are not detected in methods 0 0.001 0.01 0.1 1 10 100 1,000 10,000 proximity ligation, the assay is uniquely modular in the sense [Antigen] in 1 µL sample (pM) that new analytes can be added to an existing panel of prevalidated sequence systems without the need for reconfirming assay b 1,000 .com/nature specificity at the antibody level (S.F. and R.W.D., unpublished e data). The characteristics, reagents and performance of all six VEGF 100 proximity ligation assays are summarized in Supplementary .natur IL-4 w IL-10 Table 1 online and oligonucleotide sequences are available in IL-1α Supplementary Table 2 online. Multiplexing will become increas- TNFα 10 IL-7 ingly important as biomarker panels of multiple proteins can Signal/noise ratio 10 http://ww improve diagnostic performance . To validate the technology in biological samples, we analyzed oup 1 human plasma of 14 healthy age-matched control patients and r VEGF IL-4 IL-10 IL-1α TNFα IL-7 G Spiked protein 20 patients with untreated locally advanced pancreatic carcinoma for these six analytes in one multiplex panel and also for three Figure 2 | Multiplex proximity ligation assays. (a)Standardcurvesof additional markers in a separate higher-abundance multiplex lishing diluted analytes detected in six-plex assays. Each data point represents b panel. These first six proteins targeted for detection were not duplicate detection at the qPCR stage (error bars, 1 s.d.). (b) qPCR analysis Pu initially chosen for their relevance in pancreatic cancer but taken of individual analytes spiked into chicken plasma samples. Colored bars to represent a variety of low-abundance plasma proteins. We represent single qPCR measurements. Each x-axis category represents one added antigen. quantified each protein using standards of diluted analytes Nature (Fig. 3). The expected concentrations of these proteins in plasma 7 for healthy subjects spanned from undetectable amounts of IL-4 up 200 Optimally, the assays should use high concentrations of proximity to low-picomolar amounts of VEGF (Supplementary Table 1). © probes to promote target binding and ensure a wide dynamic range Because of each assay’s limit of detection and, more importantly, while maintaining low levels of background ligation events. The the expected abundance of the particular analyte, we did not detect background noise in proximity ligation is derived from two main sources: first, proximity probes nonspecifically binding to each other, and second, the connector oligonucleotide binding to two VEGF IL-4 IL-10 IL-1α TNFα IL-7 freely diffusing probes, enabling ligation. By probe sequence design 10 0.016 1.0 0.8 0.5 in silico, empirical experimentation and connector reconfiguration, we considerably improved the performance, permitting the detec- 0.3 8 0.8 0.4 tion of proteins in low-femtomolar concentrations in 1-mlsamples 0.012 0.6 over a five-log linear range in multiplex (Fig. 2a). A large 6 0.6 0.3 linear range is critical for multiplexed biomarker assays as proteins 0.2 vary over large concentration ranges in the blood8.Assays 0.008 0.4 using medium- to low-affinity antibodies are enhanced over 4 0.4 0.2 [Antigen] (pM) 100-fold in sensitivity by permitting the use of tenfold-higher 0.1 0.004 0.2 probe concentrations with still lower background compared to 2 0.2 0.1 previous assay configurations6. For optimization details and a comparison to previous proximity ligation configurations 0 0 0 0 0 0 and also enzyme-linked immunosorbent assay (ELISA), see Cases Cases Cases Cases Cases Cases Supplementary Figures 1 and 2 online. Controls Controls Controls Controls Controls Controls In each assay, we used either a single batch of an affinity-purified polyclonal antibody or a matched monoclonal antibody pair.
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