Article pubs.acs.org/bc
In Situ Detection of Protein Complexes and Modifications by Chemical Ligation Proximity Assay Rui Hong,* Esteban Roberts, and Christopher Bieniarz Technology and Applied Research, Ventana Medical Systems, Inc., Tucson, Arizona 85755, United States S Supporting Information *
ABSTRACT: Protein function is often regulated by protein−protein interactions and post-translational modifications. Detection of these important biological phenomena in fixed biological samples could serve as an invaluable tool in biomedical research, drug development, as well as clinical cancer diagnostics and prognostics. We report here a novel methodology which utilizes unique antibody bioconjugates capable of forming proximity induced chemical ligation to enable in situ detection of proximal targets in fixed biological samples. Using this new methodology, we demonstrate in situ visualization of various protein heterodimers/complexes and post-translational modifications such as phosphorylation and ubiquitination. This new method offers high specificity, sensitivity, flexibility, and ease of use. In addition, the assay preserves critical contextual and heterogeneity information on biomarkers in clinically relevant samples.
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INTRODUCTION Proximity assays are used to assess whether two protein targets or epitopes of interest are in close proximity. Such an assay can be used to detect protein−protein interactions (PPIs), protein dimers/complexes, and protein post-translational modifications (PTMs), all of which play critical roles in normal cellular behavior and pathogenesis. Although methods such as coimmunoprecipitation1 combined with Western blot or mass spectrometry have been widely used to study these phenomena in cell lysate or homogenized tissue samples, these methods do not preserve context and heterogeneity information on the original specimen. Several methods have also been developed to visualize protein interactions in living cells, which usually involves genetically modified tags or targets.2 A proximity assay that enables in situ detection of endogenous PPI and PTM on an individual cell basis in fixed biological samples could serve as a particularly powerful tool not only in cancer research, but also in drug development and clinical diagnostics.3 In particular, we are most interested in an in situ proximity assay for formalinfixed paraffin-embedded (FFPE) samples because nearly all the clinical tumor tissues and biopsies are fixed and preserved in this format.4 Analyzing FFPE tissues provides critical contextual information on biomarkers, which is becoming increasingly important in the era of personalized medicine.5 A few methods have been developed previously for in situ proximity detection. The most notable is the in situ proximity ligation assay (PLA) which is based on proximity-induced formation of a circular single-stranded DNA and subsequent amplification of the circular DNA by rolling circle amplification (RCA).6 Although very successful in biomedical research, the antibody conjugates and enzymes used in PLA are expensive, and require stringent storage and assay conditions to maintain their activity,7 thus inhibiting automation and wide clinical utility of PLA. © XXXX American Chemical Society
Another method for in situ detection of protein proximity is a dual binder (DB) assay, which utilizes a bispecific detection agent consisting of two Fab fragments with fast off-rate kinetics joined by a flexible linker.8 In principle, because the dual binders comprise Fab fragments with fast off-rate kinetics, the dual binders are washed off if only one of the Fab fragments is bound to its epitope. Simultaneous cooperative binding of both Fab fragments of the dual binder prevents its dissociation from the target and leads to positive staining. This approach requires development of Fab fragments with very specific binding kinetics and assay conditions with respect to the specific targets.
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RESULTS AND DISCUSSION Development of Chemical Ligation Proximity Assay. Here we report a novel chemical ligation proximity assay (CLiPA) to enable automatable in situ detection of proximal targets in fixed biological samples, including FFPE cell and tissue specimens. As depicted in Figure 1, the assay uses two modified antibodies directly or indirectly bound to the proximal targets of interest. For convenience and flexibility, antispecies antibody conjugates are used as secondary antibodies to bind to the primary antibodies of different species. Conjugate A is modified with a trifunctional molecule comprising a cleavable linker, a hapten and a first chemical ligation group CL-a. The partner Conjugate B is modified with a noncleavable molecule presenting a second chemical ligation group CL-b, which specifically reacts with CL-a to form a stable covalent bond. After binding of the antibody conjugates, a catalyst is applied to initiate the chemical ligation between CL-a and CL-b to form a stable covalent bond. A few well-established bioorthogonal Received: May 9, 2016 Revised: May 29, 2016
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DOI: 10.1021/acs.bioconjchem.6b00230 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 1. Principle and major steps of CLiPA.
reactions may be used for this purpose.9,10 The reactive groups involved are inert and stable under physiological conditions but readily form a covalent bond accelerated by a catalyst. Importantly, the ligation only occurs when the targets are in sufficient proximity11,12 because the two chemically reactive groups are confined within a few nanometers around the antibodies, depending on the linker used. It has been reported that the maximum distance of the two proximal targets detectable by PLA is about 30 nm based on the size of the antibody.6 Therefore, we expect similar detection range by CLiPA given the similar immunoassay format. A major advantage of CLiPA relative to PLA is that no special enzymes such as DNA ligase and DNA polymerase are used, and therefore it eliminates hurdles associated with these enzymes in automated staining of FFPE tissue samples. After the ligation step, the cleavable linker in Conjugate A is completely cleaved under reducing conditions. As a result, the hapten in Conjugate A is transferred to Conjugate B because of the covalent bond formed in the previous ligation step. If ligation does not occur due to insufficient target proximity, the cleaved hapten is washed off from the sample and therefore does not lead to detectable signal (bottom panel of Figure 1). Standard immunohistochemistry (IHC) staining using horseradish peroxidase (HRP) catalyzed deposition of chromogenic substrate 3,3′-diaminobenzidine (DAB) is adopted for signal development, which allows for easy visualization using brightfield microscopy.13 To further enhance the signal, a tyramide signal amplification (TSA) step can be used.14 Fluorescence based detection can also be readily achieved with a tyramide derivative of an organic dye. We adopted the well-established Huisgen 1,3-dipolar cycloaddition of alkynes to azides to form 1,4-disubsituted-1,2,3triazoles under chelated copper(I) catalysis as the chemical ligation method.15 To this end, two antibody conjugates were made as shown in Figure 2. Specifically, goat-anti-rabbit (GAR) antibody is modified with peptide Cys-HA-N3 to afford the trifunctional Conjugate A, in which the HA-tag (YPYDVPDYA) derived from human influenza hemagglutinin is used as the hapten (Supporting Information Figure S1 for synthetic scheme). The HA-tag peptide also serves as a scaffold to connect the cleavable linker (disulfide) and CL-a (azide, N3). The HA-tag peptide is made by solid phase synthesis, through which additional functionalities are readily incorporated. In
Figure 2. Structures of the antibody conjugates used in CLiPA.
addition, the cleavable disulfide linker is formed spontaneously upon reaction of the terminal cysteine residue in the peptide with GAR modified with 2-pyridyldithio functionality (Figure S2).16 The partner antibody goat-anti-mouse (GAM) is accordingly modified with a terminal alkyne group as CL-b to afford Conjugate B. We started with validation of the conjugates to ensure the required functionalities are indeed effective in the conjugates, and determination of assay conditions for each major step involved in the assay. First, the presence of the HA-tag as a detectable hapten in Conjugate A was confirmed by using a biotinylated anti-HA antibody (Figure S3). Second, the presence of acetylene reactive group in Conjugate B was confirmed by successfully performing a click reaction with biotinylated azide and subsequent detection of the presence of biotin. As expected, the click reaction only occurs in the presence of chelated Cu(I) catalyst. The reaction is quite efficient and a reaction time of 32 min was found to be sufficient (Figure S4). Third, since the read-out of proximity is based on the detection of HA-tag, the ability to completely cleave it from the antibody not engaged in the chemical ligation reaction is critical to the overall specificity of the assay. In the current format, disulfide is used as the cleavable linker and thus is cleaved using a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). To determine the conditions sufficient to achieve complete cleavage, detection of an abundant target such as protein Ki67 in tonsil combined with TSA amplification was examined. It was found that the cleavage was complete upon treatment with ∼15−20 mM DTT or TCEP for 24 min (Figure S5), and thus this condition was adopted in the following proximity assays. In addition, control experiment using an antibody labeled with a noncleavable hapten showed minimal signal reduction under this reducing B
DOI: 10.1021/acs.bioconjchem.6b00230 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry condition, indicating preservation of the detectable hapten during the cleavage step in CLiPA (Figure S6). Detection of Endogenous Protein Heterodimers/ Complexes. The complex of E-cadherin(E-cad)/p120Catenin(p120), known to be present in cells of normal and cancerous epithelial tissues,17,18 was used as a model to demonstrate the feasibility of CLiPA in FFPE specimens. We first performed CLiPA in breast cancer cells to demonstrate its specificity. As shown in Figure 3e,f, the proximity signal was
Figure 4. CLiPA of E-cad/p120 complex in normal FFPE tonsil tissue with (a) rabbit anti-E-cad only, (b) mouse anti-p120 only, (c) chelated Cu(I) added, and (d) Cu(I) excluded with otherwise identical to (c).
results strongly suggest that the antibody conjugates are specific, the cleavage of HA-tag is complete, and the chelated Cu(I) catalyzed alkyne−azide ligation is responsible for the generation of detectable signal in CLiPA. To further demonstrate the broad applicability of CLiPA, detection of DNA mismatch repair protein heterodimer MLH1/PMS2 in HeLa cell xenografts was demonstrated.21,22 Standard IHC staining of MLH1 and PMS2 is shown in Figure 5a,b, and as expected, rather uniform nuclear staining was
Figure 3. Immunocytochemistry of E-cad and p120 and detection of E-cad/p120 complex in FFPE T47D and MDA-MB-453 cells by PLA versus CLiPA. (a,b) E-cad and p120 in T47D, (c,d) E-cad and p120 in MDA-MB-453 cells, (e,f) CLiPA of E-cad/p120 complex in T47D and MDA-MB-453 cells, (f) CLiPA of E-cad/p120 complex in MDA-MB453, (g,h) PLA and CLiPA of E-cad/p120 complex in T47D cells. Figure 5. IHC of (a) PMS2 and (b) MLH1 in FFPE HeLa cell xenografts; and CLiPA of MLH1/PMS2 heterodimers with (c) chelated Cu(I) catalyst added and (d) no Cu(I) added.
only detected in T47D cells where the E-cad/p120 complex is intact, but not in MDA-MB-453 cells where the loss of E-cad leads to the translocation of p120 to the cytoplasm (Figure 3a− d).19,20 We next compared CLiPA with PLA for the detection of E-cad/p120 in T47D cells by fluorescence detection. As shown in Figure 3g,h, both methods yielded similar signal, which indicates that CLiPA is comparable with PLA. However, unlike the manual methodology of PLA, CLiPA can be performed conveniently using an automated staining system widely used in clinical anatomic pathology laboratories, thus greatly enhancing its reproducibility and throughput. As expected, both PLA and CLiPA generated no signal for Ecad/p120 in MDA-MB-453 cells (Figure S7). Detection of E-cad/p120 complex in FFPE tonsil tissues by CLiPA was further demonstrated. As shown in Figure 4, the detection of E-cad/p120 complex was achieved with high specificity and sensitivity. Importantly, positive staining was only generated when all the antibodies (primary and secondary) as well as the chelated Cu(I) catalyst were applied (Figure 4c). Control experiments by omission of one of the primary antibodies or the chelated Cu(I) catalyst lead to complete negative staining as shown in Figure 4a,b,d. These
observed in nearly all the cells. However, the CLiPA signal of MLH1/PMS2 heterodimers only exists in a subset of cells (Figure 5c), while completely negative staining was obtained with omission of the chelated Cu(I) catalyst (Figure 5d). These results suggest that although both MLH1 and PMS2 are found in the nuclei, only a subpopulation of these proteins are located at a sufficient distance to generate proximity signals, which further reflects the high proximity stringency of CLiPA. Interestingly, it has been reported that the levels of separate and functional dimerized MMR proteins fluctuate at different phases of the cell cycle in mammalian cells.23 Therefore, the signal generated by CLiPA maybe a more accurate reflection of the active status of MMR protein dimers. To the best of our knowledge, such observation has not been previously reported. In addition, the results illustrate the ability of CLiPA to preserve signal heterogeneity and contextual information in FFPE specimens. Detection of Protein Post-Translational Modifications. CLiPA can also be used to detect post-translational C
DOI: 10.1021/acs.bioconjchem.6b00230 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 6. Detection of protein PTMs by CLiPA. (a) Schematic depiction of CLiPA based detection of PTMs. (b) Detection of pTyr of EGFR in SKBr3 cells with or without EGF treatment. (c) Detection of pTyr of EGFR in clinical lung SCC and adenocarcinoma tissues in comparison with normal lung tissue.
modifications (PTMs) of proteins that are known to participate in oncogenesis including receptor tyrosine kinases (RTKs). To demonstrate this, an anti-PTM and an anti-RTK target antibody were paired as the primary antibodies in CLiPA (Figure 6a).24 In the first example, CLiPA was used to detect tyrosine phosphorylation (pTyr) of epidermal growth factor receptor (EGFR) in SKBr3 cells upon stimulation with EGF, which is known to upregulate EGFR phosphorylation. CLiPA results showed drastic increase of pTyr of EGFR in EGF stimulated cells relative to nontreated cells (Figure 6b), in agreement with the known EGF-based EGFR activation mechanism. Again, no staining was obtained with omission of the chelated Cu(I) catalyst under otherwise identical assay conditions (Figure S8). To further extend this, we demonstrated detection of pTyr of EGFR in clinical lung cancer specimens. pTyr of EGFR was detected in lung squamous cell carcinoma (SCC) and adenocarcinoma (Adeno) sections (Figure 6c) as the activation of EGFR pathway through phosphorylation has been recognized as a major mechanism and actionable target in non-small cell lung cancers.25 In comparison, the pTyr signal is minimal in normal lung tissue under the same assay conditions. Since the current assay obviates the need of a phospho-specific antibody, it could facilitate screening and identification of unknown protein phosphorylation due to the lack of a phospho-specific antibody. Another PTM known to play important biological roles is ubiquitination of proteins,26 and in situ detection of ubiquitinated proteins could further advance the understanding of their biological function and underlying mechanism.27 Since ubiquitin is an 8.5 kDa protein, it is less practical to generate specific antibodies for ubiquitinated proteins, presumably because the epitope becomes too large for sufficient antibody recognition. However, antibodies against ubiquitin generated by using full length ubiquitin as the immunogen can be paired with antibodies against specific proteins of interest to permit in situ detection of ubiquitinated proteins by CLiPA. The degradation of progesterone receptor (PR) has been known to be regulated through the ubiquitin-proteasome pathway,28 and ubiquitination of PR in MCF-7 FFPE xenograft was used as a model to demonstrate detection of ubiquitinated proteins by CLiPA.29 Again, positive detection was only achieved in the presence of chelated Cu(I) catalyst (Figure 7c). Control experiments with the omission of the chelated Cu(I) catalyst afforded totally negative stain (Figure 7d). Compared to individual detection of the two targets by IHC (Figure 7a,b), we observed a reduction
Figure 7. IHC of (a) progesterone receptor (PR) and (b) ubiquitin in MCF7 cell xenograft, and proximity assay of ubiquitinated PR by the current method (c) with chelated Cu(I) catalyst added and (d) without Cu(I) added.
of proximity signal (similar to the MLH1/PMS2 CLiPA results) which again suggests the high stringency of CLiPA in detecting close proximity rather than colocalized targets, and the capability of CLiPA to preserve signal context and heterogeneity. Overall, these results strongly support the utility of CLiPA to evaluate PTMs in preclinical and clinical FFPE specimens. It is worth noting that CLiPA is highly flexible in several aspects. First, the linker and hapten used for antibody modification can be tailored for specific targets of interest. We have used a peptide tag based hapten but other small molecule haptens can be readily incorporated to achieve the same purpose. The bioconjugation can be performed on either primary or secondary antibodies using commonly available reagents. Second, the readout can be either chromogenic or fluorescent. Third, the assays can be performed either manually or by using an automated stainer, suggesting the robustness of the method. In addition, the specificity of the assay is enhanced by the use of chelated Cu(I) catalyst as any antibody-related background is captured by negative control experiments with the omission of chelated Cu(I) under otherwise identical conditions. Therefore, we expect CLiPA to provide novel insights in many biological processes related to protein interactions and PTMs in clinically relevant tissue samples. In summary, we have developed a new enabling methodology for in situ visualization of protein complexes and posttranslational modifications in fixed biological samples, including D
DOI: 10.1021/acs.bioconjchem.6b00230 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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modified antibody was purified using a Zeba spin column, eluted in PBS (0.1 M, pH = 7.2) to provide GAM-PEG4-CCH (Conjugate B). Detection of HA-tag as a Hapten. The presence of the HA-tag peptide as a detectable hapten in Conjugate A was confirmed by IHC. IHC staining was performed on a VENTANA BenchMark XT automated slide-processing system (Ventana). Specifically, FFPE tonsil sections were first deparaffinization with EZ Prep solution (Ventana) and antigen retrieval was achieved using Cell Conditioning 1 (Ventana) (100 °C; 92 min). After through washing, ready-to-use rabbitanti-Ki67 primary antibody (clone 30−9, Ventana) was then added to the section and incubated at 37 °C for 16 min. Detection of Ki67 in tonsil was demonstrated by using GARPEG8-SS-HA-N3 as the secondary antibody, followed by antiHA-biotin (Sigma-Aldrich), SA-HRP and DAB staining using a VENTANA ultraView DAB detection kit. A typical image of the stained tissue is shown in SI Figure S3. Reactivity of Acetylene. The presence of reactive acetylene group in the conjugate of GAM-PEG4-CCH was demonstrated by performing an in situ click reaction with biotin-PEG7-N3 (Quanta Biodesign, Product #10825). IHC staining was performed on a BenchMark XT automated slideprocessing system (Ventana). Specifically, FFPE tonsil sections were first deparaffinization with EZ Prep solution (Ventana) and washed with reaction buffer. Ready-to-use mouse-antiCD20 primary antibody (clone L26, Ventana 760−2531) was added to the section and incubated at 37 °C for 16 min. After washing with reaction buffer, 100 μL of 10 μg/mL GAM-PEG4CCH (Conjugate B) was added and incubated at 37 °C for 16 min followed by thorough wash. To the section was then added 100 μL solution of 1 mM biotin-PEG7-N3, 0.6 mM CuSO4, 20 mM sodium ascorbate with two different ligand (THPTA) concentrations as indicated in Figure S4. After incubation at room temperature for 32 min, the slides were thoroughly washed. Detection of the biotin was achieved by using SA-HRP and ultraView DAB detection kit (Ventana 760−500). Determining Cleavage Conditions. To determine conditions sufficient for cleaving the HA-tag in Conjugate A, IHC staining was performed on a BenchMark XT automated slide processing system (Ventana). Briefly, FFPE tonsil sections were first deparaffinization with EZ Prep solution (Ventana) and antigen retrieval was achieved using Cell Conditioning 1 (Ventana) (100 °C; 92 min). Ready-to-use rabbit-anti-Ki67 primary antibody (clone 30−9, Ventana) was then added to the section and incubated at 37 °C for 16 min followed by incubation with GAR-PEG8-SS-HA-N3 as the secondary antibody. One hundred microliters of DTT solution of different concentration as indicated in Figure S5 was added onto the slides and incubated for 24 min. The effective concentration of the reducing reagent is usually 1/3 to 1/4 of the added solution since there is ∼300−400 μL liquid already present on the slide. The remaining HA-tag was detected by using anti-HA-biotin (Sigma-Aldrich), SA-HRP, and a tyramide signal amplification based detection using a VENTANA OptiView Amplification Kit (Ventana 860−099). Thorough wash with reaction buffer (Ventana) was performed between every step. General Procedure for the Proximity Assay Using an Automated Slide Staining System. Antibody−enzyme conjugates, detection reagents, and bulk reagents used for tissue staining were all from Ventana. IHC staining was performed on a BenchMark XT automated slide-processing system (Ventana). For all IHC staining, FFPE sections were
clinically relevant FFPE tissues. By using a bioorthogonal chemical ligation reaction rather than an enzymatic based ligation, the reagents are more readily available and costeffective, and the assay is more applicable to clinical diagnostics. We have demonstrated detection of various protein heterodimers and PTMs in FFPE tissues with high specificity and sensitivity. Combining its high specificity, versatility, ease of use, and preservation of tissue morphology and heterogeneity, this method should find many applications from basic research to potential clinical diagnostics. The assay can also be utilized as an orthogonal validation method to confirm findings from other types of experiments.
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EXPERIMENTAL PROCEDURES Materials. General chemical reagents and buffer salts were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated. SPDP-dPEG8-NHS ester was purchased from Quanta BioDesign, Ltd. (Plain City, OH, USA). Peptide Cys-HA-N3 was obtained from Bio-Synthesis, Inc. (Lewisville, TX, USA). Alkyne-PEG4-NHS ester was purchased from Click Chemistry Tools (Scottsdale, AZ, USA). Tris(3hydroxypropyltriazolylmethyl)amine (THPTA) was purchased from Sigma-Aldrich (762342). Primary antibodies used include anti-E-cadherin rabbit monoclonal antibody, clone EP700Y (Ventana Medical Systems, Inc., Tucson, AZ, USA; Ventana 760−4440); anti-p120 catenin mouse monoclonal antibody clone 98 (Ventana 790−4517); anti-EGFR rabbit monoclonal antibody clone 5B7 (Ventana 790−4347); anti-PR rabbit monoclonal antibody clone 1E2 (Ventana 790−2223); antiPMS2 rabbit mAb clone EPR3947 (Ventana 760−4531); antiMLH1 mouse mAb clone M1 (Ventana 790−4535); antiphosphotyrosine mouse mAb P-Tyr-100 (#9411, Cell Signaling Technology, Inc., Danvers, MA); anti-ubiquitin mouse mAb clone P4D1 (#3936, Cell Signaling Technology). Mouse monoclonal Anti-HA-Biotin antibody (clone HA-7) from Sigma-Aldrich (B9183−100UG) or anti-HA-Peroxidase, High Affinity from rat IgG1 from Sigma (12013819001 ROCHE) was used for the detection of HA-tag. Slides of SK-BR-3 cells untreated and epidermal growth factor (EGF)-treated (Catalog #8117) were obtained from Cell Signaling Technology, Inc. Synthesis of Conjugate A. The peptide modified antibody is synthesized following a two-step method (Figure S1 for the scheme). In the first step, the antibody (goat-anti-rabbit, GAR) was treated with SPDP-PEG8-NHS ester. In a typical reaction, 30 equiv of SPDP-PEG8-NHS ester was used and the reaction was kept at room temperature for at least 2 h. The modified GAR was purified using a Zeba spin column (Thermo Fisher Scientific Inc., Rockford, IL) eluted with PBS (pH = 7.2). The purified GAR-PEG8-SPDP was then treated with 10 equiv of peptide Cys-HA-N3 (see Supporting Information for structure) and incubated at room temperature overnight. The final conjugate GAR-PEG8-SS-HA-N3 (Conjugate A) was purified by size exclusion chromatography using Superdex 200 10/300 GL column on an AKTA purifier (GE Healthcare Bio-Sciences, Pittsburgh, PA) eluted with PBS (0.1 M, pH = 7.2). A typical chromatogram showed a peak at the end of the elution indicating the presence of 2-pyridinethione (E343 nm ∼ 8000 cm−1 M−1) as a result of the Cys-peptide conjugation to SPDP labeled antibody (Figure S2). Synthesis of Conjugate B. Goat-anti-mouse (GAM) in PBS (pH = 7.2) was treated with 15 or 30 equiv of alkynePEG4-NHS ester (see Supporting Information for structure) and incubated at room temperature for at least 2 h. The E
DOI: 10.1021/acs.bioconjchem.6b00230 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry first deparaffinization with EZ Prep solution (Ventana) and washed with reaction buffer. Antigen retrieval was carried out in Cell Conditioning 1 (Ventana) (100 °C; 92 min) and again washed with 1× reaction buffer (Ventana). Primary antibodies against the targets of interested were applied to the slide and incubated at 37 °C for 32 min. After thorough wash with reaction buffer, secondary antibody conjugates of GAR-PEG8SS-HA-N3 and GAM-PEG4-CCH (100 μL of 10 μg/mL each) were added to slides and incubated at 37 °C for 32 min. The slides were washed in reaction buffer and water before a drop of 100 μL of HEPES buffer (0.15 M, pH 7.4) was added to the slides. To initiate the azide-acetylene click reaction, 100 μL of Cu(I)/THPTA catalyst solution was added onto the slide. One working example of the Cu(I) solution contained 0.6 mM CuSO4, 3 mM THPTA, 10 mM HEPES (pH = 7.4), 40% DMSO (v/v), and 4 mM of sodium ascorbate (freshly made and added to CuSO4 immediately prior to adding to slide). The click reaction was allowed to proceed for 32 min and the slides were washed thoroughly in reaction buffer. Next, 100 μL of 50−75 mM DTT dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TECP) solution (Sigma-Aldrich) was added to the slides and incubated for 24 min. After washing, the presence of HA tag as a result of proximity induced hapten transfer was probed by the addition of anti-HA antibody. In the examples demonstrated here, a mouse monoclonal anti-HA antibody (clone HA-7) labeled with biotin (Sigma-Aldrich, B9183) was used. One hundred microliters of anti-HA-biotin at 2 μg/mL was added to each slide followed by washing and addition of 100 μL of streptavidin-horseradish peroxidase (SA-HRP) conjugate (Ventana). Alternatively, an anti-HA-Peroxidase conjugate (high affinity from rat IgG1 Roche 12013819001) was used to replace anti-HA-biotin and SA-HRP. For chromogenic based detection using, ready-to-use solutions of DAB, H2O2, and Copper from ultraView DAB detection kit were used, followed by counterstain with Hematoxylin II and Bluing (Ventana). To enhance the detection, a tyramide signal amplification scheme is adopted using an OptiView DAB detection kit (Ventana 860−099), which uses nonendogenous hapten 3-hydroxy-2-quinoxaline (HQ) coupled to tyramide. After 8 min TSA reaction and removal of excess reagent, antiHQ antibody conjugated with HRP was added and incubated for 16 min. Finally, the detection was achieved using an ultraView DAB detection kit (Ventana) with 12 min reaction followed by counterstaining with Hematoxylin II and Bluing (Ventana). The DAB staining is examined by standard bright field microscopy. Alternatively, the detection was achieved with cyanine 5tyramide conjugate (Cy5-Tyr) after the addition of SA-HRP. A TSA Plus Cyanine 5 System kit from PerkinElmer (Waltham, MA) was used. One hundred microliters of 1:50 Cyanine 5 Tyramide in 1X Plus Amplification Diluent was added to each slide and incubated for 16 min. After washing and dehydration through alcohol and xylene, the slide was mounted with VECTASHIELD HardSet Mounting Medium with DAPI (H1500, Vector Laboratories, Inc., Burlingame, CA). Fluorescence images were acquired using an Olympus BX3-CBH microscope. Proximity Ligation Assay. The proximity ligation assay was performed using Duolink In Situ PLA Probe Anti-Mouse PLUS (Sigma DUO92001) and Anti-Rabbit MINUS (Sigma DUO92005) probes with FarRed detection reagents (Sigma DUO92013). Except for the antigen retrieval step, which was performed on a BenchMark XT automated slide-processing system as previously described, all the other steps were perform
following manufactory protocol. The slides were mounted with VECTASHIELD HardSet Mounting Medium with DAPI and fluorescence images were acquired using an Olympus BX3CBH microscope.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00230. Additional experimental details and figures as indicated in the text (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare the following competing financial interest(s): All authors are employees of Ventana Medical Systems, Inc.
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ACKNOWLEDGMENTS We would like to thank Dr. Larry Morrison, Dr. Zeyu David Jiang, and Mr. Adrian Murillo for helpful discussions. REFERENCES
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DOI: 10.1021/acs.bioconjchem.6b00230 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.bioconjchem.6b00230 Bioconjugate Chem. XXXX, XXX, XXX−XXX
