Papers in Press. Published July 18, 2017 as doi:10.1373/clinchem.2017.271833 The latest version is at http://hwmaint.clinchem.aaccjnls.org/cgi/doi/10.1373/clinchem.2017.271833

Clinical Chemistry 63:9 Molecular Diagnostics and Genetics 000–000 (2017)

Analytically Sensitive Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification Tonge Ebai,1 Felipe Marques Souza de Oliveira,1 Liza Löf,1 Lotta Wik,1 Caroline Schweiger,2,4 Anders Larsson,3 Ulrich Keilholtz,4 Johannes Haybaeck,2,5 Ulf Landegren,1*† and Masood Kamali-Moghaddam1*†

BACKGROUND: Detecting at low concentrations tine instrumentation available in hospitals and research in plasma is crucial for early diagnosis. Current tech- laboratories. niques in clinical routine, such as sandwich ELISA, pro- © 2017 American Association for Clinical Chemistry vide sensitive protein detection because of a dependence on target recognition by pairs of , but detection of still lower protein concentrations is often called for. -based protein detection assays are valuable tools Proximity ligation assay with rolling circle amplification for clinical diagnostics and research applications. However, (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via the molecular complexity and wide protein concentration binding of target proteins by 3 antibodies, and signal ranges of biofluids such as plasma (1) complicate detection. amplification via rolling circle amplification (RCA) in The challenge of specific detection increases rapidly with microtiter wells, easily adapted to instrumentation in use decreasing concentrations of proteins of interest. In partic- in hospitals. ular, early detection of disease via leakage markers specifi- cally emanating from affected tissues places stringent de- mands on both the analytical specificity and sensitivity. METHODS: Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Therefore, bioassays are needed that combine high analyti- Upon target recognition these PLA probes guided oligo- cal sensitivity and specificity, as well as high precision and nucleotide ligation, followed by amplification via RCA of broad dynamic ranges. Although dedicated instrumentation circular DNA strands that formed in the reaction. The RCA is becoming available for highly sensitive protein measure- products were detected by horseradish peroxidase-labeled ment, a technology that confers improved performance us- oligonucleotides to generate colorimetric reaction products ing existing instrumentations could have broad utility. with readout in an absorbance microplate reader. Analytically sensitive protein detection assays can be designed so that binding of specifically captured proteins RESULTS: We compared detection of interleukin (IL)-4, by even single detection reagents suffices to give rise to IL-6, IL-8, p53, and growth differentiation factor 15 detectable signals. For example, sandwich immune reac- (GDF-15) by PLARCA and conventional sandwich ELISA tions confined to femtoliter reaction chambers with en- or immuno-RCA. PLARCA detected lower concentrations zymatic signal amplification have enabled digital readout of proteins and exhibited a broader dynamic range com- of single detected molecules (2–4). Similarly, eluted pared to ELISA and iRCA using the same antibodies. IL-4 products of sandwich immunoreactions have allowed and IL-6 were detected in clinical samples at femtomolar fluorophore-labeled detection complexes to pass through concentrations, considerably lower than for ELISA. a laser beam in which single fluorescent antibodies can be recorded and quantified (5, 6). Despite the single mole- CONCLUSIONS: PLARCA offers detection of lower pro- cule detection capability, the aforementioned techniques tein levels and increased dynamic ranges compared to require many molecules to be present in a sample for ELISA. The PLARCA procedure may be adapted to rou- adequate detection over background.

1 Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Upp- 75108 Uppsala, Sweden. Fax +46-18-471-4808; e-mail [email protected] and sala University, Uppsala, Sweden; 2 Charite´Comprehensive Cancer Center, University of [email protected]. Berlin, Berlin, Germany; 3 Department of Medical Sciences, Biochemical Structure and † Ulf Landegren and Masood Kamali-Moghaddam should both be considered senior authors. Function, Uppsala University, Uppsala, Sweden; 4 Institute of Pathology, Medical Uni- Received February 18, 2017; accepted May 4, 2017. versity of Graz, Graz, Austria; 5 Department of Pathology, Otto von Guericke University Previously published online at DOI: 10.1373/clinchem.2017.271833 Magdeburg, Magdeburg, Germany. © 2017 American Association for Clinical Chemistry * Address correspondence to these authors at: Uppsala University Biomedical Center, Box 815,

1

Copyright (C) 2017 by The American Association for Clinical Chemistry Alternative assays use DNA molecules as the re- PLA has also been used in (22–24) and for porter of the proteins detected. Examples of such assays enhanced Western blotting (25). are immuno-rolling circle amplification (iRCA)6 (7, 8) We report here an adaptation of the solid-phase PLA and immuno-PCR (9, 10), in which affinity reagents are protocol for protein detection using a standard microtiter conjugated to DNA molecules that can be amplified for plate reader, a format established in many clinical and the sensitive detection of labeled antibodies. research laboratories. These DNA-assisted assays have all shown analytical sensitivity in the femtomolar concentration range. Contri- Materials and Method bution to background commonly seen in conventional im- munoassays, in the form of nonspecific or ab- PATIENT SAMPLES sorbance from media, is avoided in the aforementioned The study was conducted in accordance with the Decla- assays because detectable signals can be elicited only by spe- ration of Helsinki, and it was approved by the institu- cific detection reagents. Nonetheless, other potential sources tional ethics committees of the institutions recruiting the of nonspecific signals remain in the form of absorption of patients. The Charite´approval number was EA1/069/ detection reagents to solid supports or cross-reactivity for irrel- 11, the University of Graz number was 23–015ex 10/11, evant molecules in the samples by the antibody pairs (11). and the Uppsala University number was 01/367. Patients Proximity ligation assays (PLAs) are immunoassays signed informed consent before material collection. in which pairs of antibody-conjugated DNA oligonucle- Tissue lysates were prepared from 22 primary tumors otides give rise to amplifiable reporter DNA strands only and 22 normal fresh-frozen colon tissue samples [collected upon coordinated target binding, whereas individual re- within the OncoTrack (26) project] together with zirco- agents are undetectable. The DNA ligation products that nium oxide beads and lysis buffer (50 mmol/L Tris-HCl, form can be amplified and detected by real-time PCR or pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, pH 8, 1% by means of rolling circle amplification (RCA) (12).Ina Triton X-100, 0.1% sodium deoxycholate) using a Bullit similar fashion, DNA polymerases may be used to create Blender homogenizer (Next Advance). Homogenized tis- reporter DNA strands upon pairwise binding by 2 sues were centrifuged at 13000g for 10 min at 4 °C, and the oligonucleotide-conjugated antibodies in a proximity ex- supernatants were stored at Ϫ80 °C. The total protein con- tension assay (PEA) (13). Homogenous PLA or PEA re- centration was measured (Pierce BCA Protein Assay Kit), actions lend themselves for convenient multiplex detection and the samples were diluted to 2 g/L. of proteins in single microliter samples with no need for Blood samples from 25 patients with prostate can- washes, and multiplex PEA are commercially available. The cer, ages 34–70 years, and from 24 age-matched healthy analytical specificity may be extended, and large sample vol- male controls (see Table 1 in the Data Supplement that umes can be used to enhance analytical sensitivity by first accompanies the online version of this article at http:// capturing target proteins on a solid support in simplex www.clinchem.org/content/vol63/issue9) were collected (12, 14) or multiplex (15, 16) before pairs of PLA probes in Vacutainer tubes containing K2-EDTA (Cat. #454410, are introduced, followed by washes to remove excess re- Becton Dickinson). The tubes were centrifuged at 1500g for agents, ligation, and then detection via real-time PCR. 10 min at room temperature, and the plasma was transferred A variant of PLA, in situ PLA, is used to image to new tubes, which were stored at Ϫ80 °C. proteins, protein–protein interactions, and posttransla- tional modified proteins in situ by microscopy (17–21). ANTIBODIES, , AND OLIGONUCLEOTIDES For such localized detection reactions, amplification The antigens and their cognate affinity-purified poly- through RCA of DNA circles that form via ligation in a clonal antibodies are presented in Table 2 in the online target-dependent manner is used in place of PCR. Each Data Supplement, and the oligonucleotide systems for RCA product is a concatamer of typically hundreds of proximity ligation assay with rolling circle amplification complements of the DNA circle that templated its synthesis, (PLARCA) and iRCA are listed in Table 3 in the online collapsed into a submicrometer ball of single-stranded Data Supplement. DNA. These amplification products can be detected using fluorophore- or enzyme-labeled oligonucleotide probes, hy- PREPARATION OF PLA PROBES bridizing to their repeated sequence, and are visible as bright PLA probes were prepared by covalently conjugating fluorescent or colored spots by microscopy. RCA-mediated oligonucleotides to antibodies. Next, 25 mmol/L diben- zylcyclooctyne N-hydroxysuccinimide ester (Jena Biosci- ence) was dissolved freshly in 4 mmol/L DMSO (Sigma- Aldrich) in a 25-fold molar excess of dibenzylcyclooctyne 6 Nonstandard abbreviations: iRCA, immuno-rolling circle amplification; PLA, proximity N-hydroxysuccinimide ester. Antibodies were diluted in ligation assay; RCA, rolling circle amplification; PLARCA, proximity ligation assay with ϫ ␮ ␮ rolling circle amplification; GDF, growth differentiation factor; LOD, limit of detection; 1 PBS at 2 g/ L, and a 33.3-fold molar excess of the LLOQ, lower limits of quantification. freshly prepared dibenzylcyclooctyne was added and in-

2 Clinical Chemistry 63:9 (2017) Sensitive Protein Detection Using PLARCA

cubated at room temperature for 30 min in the dark. The activated antibodies were purified using ZebaTM Spin A Desalting Columns, 7K MWCO, 0.5 mL (Thermo Sci- entific), having been equilibrated with 1ϫ PBS as di- rected by the manufacturer. Purified antibodies were di- vided in 2 aliquots of 1 ␮g/␮L and mixed with a 2.5-fold I II III IV V molar excess of either the S3 primer or the S3 2Ј-O- methyl RNA base-blocked azide-modified oligonucleo- tides, and incubated overnight at 4 °C. B

PHOSPHORYLATION OF OLIGONUCLEOTIDES The DNA (stock concentration 100 ␮mol/L) oligonu- cleotides were mixed to a final concentration of 10 I II III IV V ␮ ϫ mol/L in 1 T4 polynucleotide kinase reaction buffer ␮ A (Fermentas) with 0.25 U/ LT4 polynucleotide kinase C (Fermentas), 10 mmol/L ATP, and incubated for 30 min at 37 °C, followed by inactivation at 75 °C for 10 min in a thermocycler. The phosphorylated oligonucleotides were immediately placed on ice before being stored at Ϫ 20 °C for further use. The oligonucleotides can also be PLARCA iRCA ELISA ordered with 5Ј phosphates as required for their ligation.

Fig. 1. Schematic descriptions and comparison of PLARCA, PLA WITH ABSORBANCE READOUT Specific antibodies, 200–800 ng added per well, were iRCA, and standard sandwich ELISA. coated in a 96-well microtiter plate in coating buffer (100 Schematic description of the PLARCA assay (A). Antibodies are im- mmol/L carbonate buffer, pH 9.6) at 4 °C overnight be- mobilized via hydrophobic interaction in wells of microtiter plates, fore the wells were blocked with 200 ␮L of 1% BSA in followed by washes and blocking of nonspecific binding (I). Bound 1ϫ PBSfor1hatroom temperature. The plates were antibodies are used to capture target proteins from added samples, then rinsed in washing buffer (1ϫ PBS, 0.01% BSA, and followedbywashestoremoveunboundsamplecomponents.Next,a 0.05% Tween 20), and the top surface was blotted using pair of PLA probes (antibody-DNA conjugates) is added that can rec- tissue paper after each step in the assay. Dilution series of ognizecapturedtargetmolecules,followedbywashes(II).DNAoligo- proteins were prepared in PLA buffer or 50% or 10% nucleotides are then added along with a ligase allowing the oligonu- chicken serum (Invitrogen) as indicated for the individ- cleotides to be ligated into DNA circles, templated by an ual experiments. All dilution series contained negative oligonucleotide on one of the PLA probes (III). A DNA polymerase is controls with no protein added to determine the back- added to initiate replication of the DNA circles via RCA, primed from ground. The assays were initiated by incubating 100 ␮L that antibody-conjugated oligonucleotide that did not template liga- samples in antibody-coated microtiter wells for1hat tion(IV).Finally,theRCAproductsaredetectedbyaddinghorseradish room temperature on a shaker (80 rpm) followed by 3 peroxidase-labeled oligonucleotides to the RCA product, followed by washes. The PLA probes were diluted in PLA buffer washeswithadditionofthe3`3`,5`5`-tetramethylbenzidinesubstrate (0.1% BSA; New England BioLabs), 0.05% Tween (V). Schematic description of iRCA (B). Same as under (A) except that a (Sigma-Aldrich), 0.1 ␮g/␮L salmon sperm DNA (Invit- single oligonucleotide-modified antibody is used both to template rogen), 100 nmol/L goat IgG (Sigma-Aldrich), and 5 ligation and to prime RCA. Schematic comparison of PLARCA, iRCA, mmol/L EDTA (1ϫ PBS) to a final concentration of 1 and a standard sandwich ELISA (C). nmol/L, from which 100 ␮L was added to each well, followed by incubation for1hatroom temperature on a shaker. The wells were washed again, and 100 ␮Lofa followed by incubation for 1–1.5 h at 37 °C and then by ␮ ϫ ligation mix (0.5 U/ LT4 DNA ligase; New England washes. A 2 hybridization buffer containing 40% for- ϫ ϫ BioLabs) in 1 T4 ligase buffer without dithiothreitol mamide and 4 sodium saline citrate (0.15 mol/L NaCl but with 0.25 g/L BSA, 0.5 mmol/L ATP, and 200 and 0.015 mol/L sodium citrate ϭ 1ϫ SSC) was pre- nmol/L of the 2 circle-forming oligonucleotides, was pared and stored in the dark. Then, 100 ␮L of detection added into each well and incubated for 30 min at 37 °C mix (50 pmol/L horseradish peroxidase-labeled detection followed by washes. Next, 100 ␮L of RCA mix [0.5 oligonucleotide, 1ϫ hybridization buffer) was added and U/␮L Phi29 polymerase (Fermentas) in 1ϫ Phi29 poly- incubated for 30 min at 37 °C, followed by washes. Fi- merase buffer (Fermentas) with 0.25 g/L BSA, 0.4 nally, 50 ␮L of the horseradish peroxidase substrate, Ј Ј mmol/L dNTPs (Fermentas), and ddH2O] was added, 3,3 ,5,5 -tetramethylbenzidine (Sigma-Aldrich), equili-

Clinical Chemistry 63:9 (2017) 3 [IL-2] (pmol/L) [IL-6] (pmol/L) 0.0 1E-05 0.0001 0.001 0.01 0.1 1 10 100 0.0 0.001 0.01 0.1 1.0 10 100 1000

Buffer Buffer 10% chicken serum 10% chicken serum 50% chicken serum 3 3 50% chicken serum

0.3 0.3 OD (450 nm) OD (450 nm)

0.03 0.03

0.0 0.02 0.2 2.0 20 200 2000 20000 0.0 1.5 15 150 0.015 0.15 1500 1.5E-05 [IL-6] (pg/mL) 1.5E-04 [IL-2] (pg/mL)

[IL-8] (pmol/L) [VEGF] (pmol/L) 0.0 1E-04 1E-03 0.01 0.1 1 10 100 1000 0.0 1E-05 1E-04 0.001 0.01 0.1 1.0 10 100 1000

Buffer Buffer 10% chicken serum 10% chicken serum 50% chicken serum 50% chicken serum 3 3

0.3 0.3 OD (450 nm) OD (450 nm)

0.03 0.03

0.0 0.0008 0.008 0.08 0.8 8.0 80 800 8000 0.0 4.2E-05 0.0042 0.042 0.42 4.2 42 420 4200 [IL-8] (pg/mL) [VEGF] (pg/mL)

Fig. 2. Validation of PLARCA performance in complex biological samples. Comparison of results from assays for interleukin (IL)-2, IL-6, IL-8, and vascular endothelial growth factor in buffer (blue) or 10% (red) or 50% (green) chicken serum. The y axes show optical density (OD) measured at 450 nm, whereas the x axes show protein concentrations in molarity (top) and weight per volume (bottom). The means of triplicate measurements are shown with SDs indicated with error bars. brated at room temperature, was added to each well, in- SANDWICH ELISA cubated for 10 min at room temperature, and then The R&D Quantikine kits for IL-4, IL-6, and growth stopped with an equal volume of 2 mol/L H2SO4 or differentiation factor (GDF-15) were used according to H2PO4. The colored product was then measured using the manufacturer’s instructions (see Table 2 in the online an absorbance plate reader within 30 min at 450 nm and Data Supplement). The proteins were spiked in 10% and with absorbance at 650 nm as reference. 50% calibrator diluent to ensure fair comparison. iRCA DATA ANALYSIS The assay was performed similarly to the PLARCA de- The absorbance data were recorded using a Safire II mi- scribed above with the exception that only one PLA con- crotiter plate reader, and the optical densities (OD 450 jugate, with the S3 primer, was used to template the nm) were exported and further analyzed with Microsoft formation of a DNA circle via a single ligation, and to Excel and StatPlus LE. A 4-parametric logistic linear re- prime RCA, as illustrated (Fig. 1B). gression was used to calculate the limits of detection

4 Clinical Chemistry 63:9 (2017) Sensitive Protein Detection Using PLARCA

Table 1. Assay summary of the analytical performance of PLARCA of IL-2, IL-6, IL-8, and VEGF measured in buffer and 10% and 50% serum.a

IL-2 IL-6 IL-8 VEGF

Buffer 10% 50% Buffer 10% 50% Buffer 10% 50% Buffer 10% 50% LLOQ (pg/mL) 0.333 0.794 0.968 0.910 0.794 0.4753 0.413 0.492 0.471 0.042 0.079 0.095 LOD (pg/mL) 0.031 0.145 0.045 0.074 0.200 0.133 0.131 0.247 0.236 0.047 0.081 0.102 Intraassay CV (%) 8.5 5.0 8.0 4.3 11.5 9.8 8.3 10.0 7.6 5.3 6.3 7.7 (n=6) Interassay CV (%) 9.9 6.8 14.0 10.8 18.2 14.1 15.1 24.2 21.2 7.9 13.6 9.3 (n=6)

a IL, interleukin; VEGF, vascular endothelial growth factor; LLOQ, lower limits of quantification.

(LOD) and the lower limits of quantification (LLOQ). protein preparations were diluted over a million-fold Statistical significances of measurements for the patient concentration range in buffer or in 10% and 50% and control samples were calculated using the Wilcoxon chicken serum before analysis (Fig. 2) to represent a com- signed rank test. To calculate the P values, all the data plex biological matrix. With the increase in matrix com- points below the LODs of the assays were put at the plexity from buffer to chicken serum, we observed a LODs. The LOD values were calculated as the protein slightly increased background for some of the analytes concentrations on the fitted curves that corresponded to (Fig. 2) but with limited effect on the LOD and LLOQ OD values representing the average OD of 3 negative of the assays (Table 1). control samples plus 3 standard deviations of those val- The analytical characteristics of the assays are sum- ues. Similarly, the LLOQ were calculated as the protein marized in Table 1, with the LOD defined as the con- concentrations on the fitted curves corresponding to the centration of protein detected at 3 SDs over the back- average OD background of 3 negative control samples ground. We compared the performance of PLARCA plus 10 standard deviations. with conventional sandwich for IL-4, IL6, and GDF-15 (Fig. 3A), and with iRCA for IL-8 and p53 (Fig. Results 3B), all spiked in 50% chicken serum. Monoclonal anti- bodies, used as capture reagents for PLARCA, were im- The method described here, which we term PLARCA, relies mobilized in microtiter wells overnight in coating buffer. on target protein capture by immobilized antibodies, fol- PLA probes were prepared from polyclonal antibody lowed by detection of the protein via pairs of antibody– preparations as described in Materials and Methods. As- DNA conjugates (PLA probes) that give rise to circular say diluent, substrate, and stop solution from the ELISA DNA strands in the same manner as for in situ PLA with kit were used to compare PLARCA, iRCA, and ELISA. signal amplification via RCA. The amount of RCA prod- ucts is measured by hybridization with DNA probes con- We compared the performance of PLARCA with that of jugated to horseradish peroxidase, acting on the substrate ELISA kits from R&D Systems for detection of IL4, 3,3Ј,5,5Ј-tetramethylbenzidine to generate colorimetric IL-6, and GDF-15 (Fig. 3). Although ELISA took less reaction products that are read out with an absorbance time than PLARCA, PLARCA in serum demonstrated Ͼ microplate reader (Fig. 1). 86-, 15-, and 2-fold increased analytical sensitivity for We prepared PLA probes by covalently coupling an- detection of IL-4, IL-6, and GDF-15, respectively, com- tibodies to DNA oligonucleotides using click chemistry pared with ELISA. PLARCA offered 2 orders of magni- through residues introduced in the antibodies via a bi- tude greater dynamic range for IL-4 and IL-6 and 1 functional dibenzylcyclooctyne N-hydroxysuccinimide order of magnitude greater dynamic range for GDF-15 ester cross-linker. The quality of each batch of conjugates (Table 2). The mean %CV for ELISA was about 5% was validated with agarose gel electrophoresis for each for the intraassay variation compared with about 10% new assay (see Fig. 1 in the online Data Supplement). for PLARCA. Two designs of DNA oligonucleotides coupled to We also compared PLARCA with iRCA assays for the antibody were tested to identify sequences with opti- IL-8 and p53. PLARCA showed Ͼ3.5- and 3-fold mal signal-to-noise ratio for use in the experiments de- increased analytical sensitivity for detection compared scribed here. To investigate the assay sensitivity, pure with iRCA for IL-8 and p53, respectively (Fig. 3B and IL-2, IL-6, IL-8, and vascular endothelial growth factor Table 2).

Clinical Chemistry 63:9 (2017) 5 AB[GDF-15] (pmol/L) 0.0 0.071 0.71 7.1 71.0 710 10 [IL-8] (pmol/L) 0.0 1E-04 0.001 0.01 0.1 1 10010 1000 ELISA 3 LOD iRCA PLARCA LOD LOD PLARCA 1 LOD 0.3 PLARCA PLARCA OD (450 nm) ELISA iRCA OD 450 nm 0.01 0.03

0.001 0.003 0.0 1 10 100 1000 10000 0.0 0.0008 0.008 0.08 0.8 8 80 800 8000 [IL-8] (pg/mL) [GDF-15] (pg/mL)

[IL-4] (pmol/L) [p53] (pmol/L) 0.0 0.0007 0.067 0.67 6.7 67 670 1E-050.0 1E-04 0.010.001 1.00.1 10 100 10 10

ELISA LOD iRCA PLARCA LOD LOD 1 PLARCA 1 LOD PLARCA ELISA PLARCA iRCA OD (450 nm) OD (450 nm) 0.1 0.1

0.01 0.01 0.0 0.001 0.1 10 1001000 10000 0.0 0.53 5.3 53 00 [IL-4] (pg/mL) 5.3E-035.3E-02 0.053 530 53 [p53] (pg/mL) [IL-6] (pmol/L) 0.0 0.049 0.49 4.9 49

10 ELISA LOD PLARCA LOD

1

PLARCA

OD (450 nm) ELISA

0.1

0.01 0.0 1 10 100 1000 [IL-6] (pg/mL)

Fig. 3. Performance of PLARCA vs ELISA and iRCA. Comparison of PLARCA (squares) and ELISA (diamonds) for measuring levels of interleukin (IL)-4, IL-6, and GDF-15 (A). Comparison between PLARCA (squares) and iRCA (diamonds) for measuring concentrations of IL-8 and p53 (B). All analytes for PLARCA, iRCA, and ELISA were spiked in 50% calibrator diluent for serum. The means of triplicate measurements are shown with SDs indicated with error bars.

6 Clinical Chemistry 63:9 (2017) Sensitive Protein Detection Using PLARCA

Table 2. Comparison of LOD, LLOQ, dynamic range, and time for PLARCA in measurements of IL-4, GDF-15, and IL-6 with ELISA and of IL-8 and p53 with iRCA.a

IL-4 GDF-15 IL-6 IL-8 p53

PLARCA ELISA PLARCA ELISA PLARCA ELISA PLARCA iRCA PLARCA iRCA LOD (pg/mL) 0.099 8.47 0.899 1.37 0.089 1.373 0.041 0.161 0.020 0.143 LLOQ (pg/mL) 0.129 10.59 1.43 1.85 0.1928 9.03 0.237 0.902 0.765 1.617 Dynamic range 104 102 103 102 104 102 105 104 105 104 Time (h) 6.25 4.5 6.25 4.5 6.25 4.5 6.25 6.26 6.27 6.28

a LLOQ, lower limits of quantification; IL, interleukin. All assays were performed in 50% serum.

We further compared the performance of PLARCA measure proteins at very low concentrations and over with our previously described microparticle-based solid- broad detection ranges, permitting detection of promis- phase PLA protocol (14) for IL-6 and vascular endothe- ing protein biomarkers in plasma using routine labora- lial growth factor in 10% chicken serum, showing com- tory equipment. The requirement for detection by sets of parable analytical sensitivity for detection and dynamic 3 affinity reagents in PLARCA can serve to enhance the ranges (see Fig. 2 in the online Data Supplement). analytical specificity of detection compared with that of Finally, to evaluate the performance of PLARCA in sandwich ELISA and/or iRCA by reducing risks of cross- clinical samples, we measured concentrations of IL-6 in reactivity, while the solid supports also permit analysis of tissue lysates prepared from tumor tissues and surround- large sample volumes and washes to remove excess re- ing healthy intestinal samples from 22 patients with co- agents. This is in contrast to homogenous PLA and PEA lon cancer (see Table 1 in the online Data Supplement). that are typically limited to sample volumes of 1 ␮L PLARCA distinguished IL-6 concentrations between ly- because of the need to reduce background via dilution of sates of tumor and nontumor tissues with a P value the reactions before the enzymatic step. PLA measure- Ͻ0.001. Furthermore, detectable IL-6 concentrations ments also reduce risks of background signals because of were found in most of the tumor and control tissue ly- nonspecifically bound detection reagents, as only pairs of sates using PLARCA, whereas the concentrations of IL-6 PLA probes can produce detection signals, whereas single in most of the samples were below the detection limit for nonspecifically bound detection reagents are undetect- ELISA (Fig. 4A). In addition, we also measured the con- able. The requirement for specific DNA hybridization centrations of IL-4 and IL-6 in plasma samples from 25 and ligation ensures that only cognate pairs of detection patients with prostate cancer and 24 samples from reagents give rise to the signal. This has proven highly healthy controls (see Table 1 in the online Data Supple- useful when more PLA probe pairs are added in multiplex ment). ELISA recorded detectable concentrations of IL-4 reactions (27, 28). Across all investigated proteins, (Fig. 4B) and IL-6 (Fig. 4C) in 16% and 32% of the PLARCA consistently achieved low detection limits, patients, respectively, whereas PLARCA successfully de- broad dynamic ranges, and high precision. Moreover, tected protein concentrations in Ͼ80% and 84% of the interfering substances from plasma had only a limited samples, respectively. influence on the results. PLARCA had intraassay CV% of about 5% to 11%, considerably less than our previ- Discussion ously reported solid-phase PLA with real-time PCR read- out for some assays (14). We report here a new format for PLA, suitable for detect- Compared with conventional ELISA and iRCA, ing proteins in low femtomolar concentrations in com- PLARCA offered Ͼ100-fold greater analytical sensitivity plex media, using antibody-conjugated oligonucleotides for detection of some cytokines. In samples from patients (12) that jointly but not individually are able to elicit with colon cancer, PLARCA better differentiated be- signal amplification via RCA (17). The assays are per- tween lysates prepared from tumor tissues and surround- formed in standard microtiter wells with removal by ing healthy tissues compared with ELISA. PLARCA washes of excess reagents and sample components that could also detect proteins at concentrations that were could interfere with the assay, followed by recording of below the detection limits for ELISA. Recently an the absorbance in a standard microplate reader. Our re- ELISA-PLA protocol was reported for detection of post- sults show that PLARCA has the potential to detect and translational modifications of proteins by directly adapt-

Clinical Chemistry 63:9 (2017) 7 ing the in situ PLA protocol to bind capture and detec- A P < 0.0001 1000 tion antibodies in microtiter plates, for which higher sensitivity than ELISA was demonstrated (29). In con- 100 trast to our present study, target molecules were recog- P = 0.108 10 nized by only 2, rather than 3, antibodies, limiting the LOD 1 analytical specificity of detection. PLARCA assays can theoretically be easily estab- 0.1 [IL-6] (pg/mL) LOD lished for individual proteins, provided high-quality af- 0.01 finity reagents are available. Protein–DNA conjugation is now a routine procedure using conjugation chemistries Cancer tissue Healthy tissue Cancer tissue Healthy tissue such as the click chemistry used in this study. Assays with PLARCA ELISA the 3 affinity reagents required for each target protein can

B 1000 P < 0.0001 be established using combinations of monoclonal and polyclonal antibodies as described herein. Alternatively,

100 we have previously shown that a single polyclonal anti- body preparation raised against all or a substantial por-

10 P = 0.348 tion of the target protein can regularly be divided in 3 aliquots and used for a capture antibody preparation and 1 LOD 2 PLARCA probes. As demonstrated for PLARCA here [IL-4] (pg/mL) and previously for solid-phase PLA (28), these triple- 0.1 binder assays work well for proteins with sizes ranging LOD from as little as 8.6 kDa (IL-8) to 43.7 kDa (p53). 0.01 In conclusion, we have developed a new assay format for protein detection called PLARCA, which offers an Patients Controls Patients Controls opportunity to detect proteins at low femtomolar con-

PLARCA ELISA centrations in buffer and in biological samples. PLARCA could be implemented in any laboratory where micro- C 1000 plate readers are in use. P < 0.001 100

10 Author Contributions: All authors confirmed they have contributed to P = 0.521 the intellectual content of this paper and have met the following 3 require- 1 ments: (a) significant contributions to the conception and design, acquisi- tion of data, or analysis and interpretation of data; (b) drafting or revising [IL-6] (pg/mL) LOD 0.1 the article for intellectual content; and (c) final approval of the published article.

0.01 LOD Authors’ Disclosures or Potential Conflicts of Interest: Upon man- uscript submission, all authors completed the author disclosure form. Dis- closures and/or potential conflicts of interest: Patients Controls Patients Controls Employment or Leadership: U. Landegren, Olink Proteomics. PLARCA ELISA Consultant or Advisory Role: U. Landegren, Olink Proteomics. Stock Ownership: U. Landegren, Olink Proteomics. Fig. 4. Measurement of cytokine concentrations in colorec- Honoraria: None declared. tal and prostate cancer. Research Funding: U. Keilholtz, the Innovative Medicines Initiative Joint Undertaking under grant agreement N° 115234 (OncoTrack); J. Measurement of interleukin (IL)-6 PLARCA and ELISA in 22 tissue ly- Haybaeck, the Innovative Medicines Initiative Joint Undertaking un- sates from colon cancer tissue and 22 normal colonic samples from der grant agreement N° 115234 (OncoTrack); U. Landegren, The the same patients (A). Measurement of IL-4 (B) and of IL-6 (C) with European Research Council under the European Union’s Seventh Framework Programme (FP/2007–2013) Grant Agreement N° PLARCA and ELISA in plasma samples from 25 prostate cancer pa- 294409 (ProteinSeq), n° 264737 (BIOMAX), N° 313010 (BBMRI- tients and 24 healthy control subjects. PLARCA and ELISA LODs were LPC), The Swedish Research Council, IngaBritt and Arne Lundbergs calculated from the standards in Table 2 for IL-4 and IL-6. The age Forskningsstiftelse, the Knut and Alice Wallenberg Foundation, the rangesforthesepatientsandcontrolsubjectsareillustratedinTable2 Innovative Medicines Initiative Joint Undertaking under grant agree- in the online Data Supplement. Each dot represents the mean of a ment N° 115234 (OncoTrack); M. Kamali-Moghaddam, N° 316929 (GastricGlycoExplorer), The Swedish Research Council, IngaBritt and triplicate. P values were calculated using a 2-sample Wilcoxon rank Arne Lundbergs Forskningsstiftelse. sum test. Expert Testimony: None declared. Patents: None declared.

8 Clinical Chemistry 63:9 (2017) Sensitive Protein Detection Using PLARCA

Other Remuneration: U. Landegren, a founder and shareholder of Role of Sponsor: The funding organizations played no role in the Olink Proteomics and Olink Bioscience, having rights to the proximity design of study, choice of enrolled patients, review and interpretation of ligation technology. data, and final approval of manuscript. References

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