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Rapid homogeneous immunoassay for cardiac troponin I using switchable lanthanide luminescence
Biosensors and Bioelectronics 62 (2014) 201–207
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Rapid homogeneous immunoassay for cardiac troponin I using switchable lanthanide luminescence Henna Päkkilä n, Eeva Malmi, Satu Lahtinen, Tero Soukka Department of Biochemistry/Biotechnology, Tykistökatu 6A, 6th Floor, FI-20520 Turku, Finland
art ic l e i nf o
a b s t r a c t
Article history: Received 9 May 2014 Received in revised form 18 June 2014 Accepted 22 June 2014 Available online 25 June 2014
Homogeneous assays are advantageous because of their simplicity and rapid kinetics but typically their performance is severely compromised compared to heterogeneous assay formats. Here, we report a homogeneous immunoassay utilizing switchable lanthanide luminescence for detection and sitespecifically labeled recombinant antibody fragments as binders to improve the assay performance. Switchable lanthanide luminescence enabled elimination of assay background due to division of the luminescent lanthanide chelate into two non-luminescent label moieties. Simultaneous biomolecular recognition of model analyte cardiac troponin I by two antibody fragments brought the label moieties together and resulted in self-assembly of luminescent mixed chelate complex. The assay was very rapid as maximal signal-to-background ratios were achieved already after 6 min of incubation. Additionally, the limit of detection was 0.38 ng/mL (16 pM), which was comparable to the limit of detection for the heterogeneous reference assay based on the same binders (0.26 ng/mL or 11 pM). This is the first study to apply switchable lanthanide luminescence in immunoassays and demonstrates the versatile potential of the technology for rapid and sensitive homogeneous assays. & 2014 Elsevier B.V. All rights reserved.
Keywords: Lanthanide chelate Immunoassay Homogeneous detection Bioconjugation Cardiac troponin I
1. Introduction Homogeneous assay formats are well suited for point-of-care tests because they are simple to perform and have fast binding kinetics. However, matrix effects and high background signal are common drawbacks of the homogeneous assays. The challenge in using antibodies as binders in especially fluorescence-based homogeneous protein detection is their large size, which may take the labels too far apart in the formed immunocomplex to allow signal generation. Moreover, varying labeling degrees and random positions of the labels in the antibodies hamper the signal generation. The problems related to the use of antibodies in homogeneous assays have been avoided by using oligonucleotides attached to antibodies to enable nucleic acid amplification, such as in proximity-ligation assays (Darmanis et al., 2010; Fredriksson et al., 2002; Hammond et al., 2012) and binding-induced DNAassembly (Zhang et al., 2012). In these applications, the length of oligonucleotides can be adjusted depending on the size of an analyte to enable hybridization and following nucleic acid amplification. The use of direct fluorescence detection, such as Förster resonance energy transfer (FRET), avoids the need for timeconsuming nucleic acid amplification. Also in the FRET-based n
Corresponding author. Tel.: þ 358 2 333 8089. E-mail addresses: henna.pakkila@utu.fi (H. Päkkilä), esmalm@utu.fi (E. Malmi), selaht@utu.fi (S. Lahtinen), tejoso@utu.fi (T. Soukka). http://dx.doi.org/10.1016/j.bios.2014.06.042 0956-5663/& 2014 Elsevier B.V. All rights reserved.
assays, oligonucleotides have been used to assist in bringing the labels into close proximity to enable the generation of fluorescence signal (Heyduk et al., 2008, 2010). Lanthanide ions form brightly luminescent complexes when chelated to organic ligands containing light-harvesting chromophores. The ligand allows efficient excitation of the lanthanide ion and shields it from the quenching effects of water. The major advantage of lanthanide chelates is their long luminescence lifetime in millisecond range, which enables the use of timeresolved measurement to eliminate the short lifetime autofluorescence background. Furthermore, narrow emission bands and large Stokes shift are characteristic for lanthanide labels. Additional advantage of lanthanide chelate labels over conventional luminescent labels is that no self-quenching is observed enabling high labeling degree (Bünzli, 2010; Hemmilä et al., 1984). Switchable lanthanide luminescence is a novel label technology, which enables sensitive and specific homogeneous binding assays (Karhunen et al., 2010, 2011). The luminescent lanthanide chelate is split into two non-luminescent label moieties, which are covalently attached to two separate biomolecular binders. The binders recognize the analyte bringing the label moieties into close proximity, which increases the local concentration of the label moieties and enables lanthanide chelate self-assembly. Without the analyte, the label moieties remain apart and no luminescence signal is detected. The obtained signal is very specific to the
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presence of the analyte because two separate binding events are required for signal generation. The applicability of switchable lanthanide luminescence has been demonstrated in nucleic acid hybridization assays (Karhunen et al., 2010, 2013; Kitamura et al., 2008; Oser and Valet, 1990; Wang et al., 2001) and the principle has also been applied to nucleic acid amplification (Lehmusvuori et al., 2012, 2013) as well as to ligand and aptamer-based protein detection (Karhunen et al., 2011; Päkkilä et al., 2013). In this study, switchable lanthanide luminescence was used for the first time together with antibody binders. The label moieties were attached to fragment antigen binding portions of antibodies (Fabs) via oligonucleotides allowing direct detection and enabling rapid assay, which is more applicable for point-ofcare tests than those requiring nucleic acid amplification. The reporter oligonucleotides carrying the label moieties were hybridized to linker oligonucleotides that were covalently coupled to Fabs. The high background signal inherent to FRET-based assays and organic fluorophores was avoided by the use of long-lifetime labels and time-resolved luminescence measurement. The use of recombinant Fabs enabled site-specific coupling of linker oligonucleotides and prevented oligonucleotides from interfering with analyte recognition (Fig. 1). Replacing intact antibodies with Fabs has been shown to improve the detection limit of heterogeneous assays because more dense and well-oriented binding surface is obtained resulting in improved binding capacity and higher signal levels (Ylikotila et al., 2006). In addition, their use reduces the risk of interference caused by human anti-mouse antibodies in clinical samples (Hyytiä et al., 2013). Cardiac troponin I (cTnI) was used as a model analyte to demonstrate the potential of the assay concept. CTnI has high cardiac tissue specificity and clinical sensitivity, which makes it an ideal biomarker for the diagnosis of acute myocardial infarction (MI) (Thygesen et al., 2012). CTnI assays should have short turn-around times to enable early diagnosis and rapid treatment. While novel central laboratorybased tests are high-sensitivity assays, point-of-care tests with equal performance are still lacking (Lee-Lewandrowski et al., 2011).
Table 1 Oligonucleotide sequences. Name
Sequencea
Linker A Linker B Reporter Reporter Reporter Reporter Reporter Reporter
5′-#TTT TTT TTT TTT TTT TTT CCA GTT GCA GCA CGG-3′ 5′-GGA AGA CTC GAC AGC TTT TTT TTT TTT TTT TTT#-3′ 5′-*GCG TGG TTT TTC CGT GCT GCA ACT GG-3′ 5′-GCT GTC GAG TCT TCC TTT TTC CAC GCT*-3′ 5′-*GCG TGT GTT TTT CCG TGC TGC AAC TGG-3′ 5′-GCT GTC GAG TCT TCC TTT TTC ACA CGC T*-3′ 5′-*TGC GTG TGT TTT TCC GTG CTG CAA CTG G-3′ 5′-GCT GTC GAG TCT TCC TTT TTC ACA CGC AT*-3′
A6 B6 A7 B7 A8 B8
# Terminal maleimide modification, * terminal amino modification with six carbon aliphatic spacer. a Bolded nucleotides denote the reporter stem and underlined nucleotides the complementary region between linkers and reporters.
2. Materials and methods 2.1. Materials Human cardiac troponin complex was purchased from Hytest (Turku, Finland) and oligonucleotides (Table 1) were from Biomers. net (Ulm, Germany). Fab 4C2 and Fab MF4 used in homogeneous and heterogeneous assays were produced and biotinylated as previously described (Ylikotila et al., 2006). Assay buffer (50 mM Tris–HCl, pH 7.75 containing 150 mM NaCl, 0.05% (w/v) NaN3, 0.01% (w/v) Tween 40, 0.05% (w/v) bovine γ-globulin, 20 μM diethylenetriamine pentacetic acid and 0.5% (w/v) bovine serum albumin) and wash solution (5 mM Tris–HCl, pH 7.75 containing 150 mM NaCl, 0.005% (w/v) Tween 20 and 0.1% (w/v) Germall II) were from Kaivogen Oy (Turku, Finland) as well as the low fluorescence streptavidin-coated microplates used in heterogeneous reference assay. The low fluorescence 96-well MaxiSorp microtitration plates were from Nunc (Roskilde, Denmark). Victor Multilabel Plate Reader X4 used to measure the time-resolved luminescence was purchased from Wallac/Perkin-Elmer Life and Analytical Sciences (Wellesley, MA). Time-resolved Eu(III) luminescence intensities were measured using default factory-provided filters for 340 nm excitation and 615 nm emission. For the characterization of labeled assay components, both delay time and window time were 400 ms, but in the measurements of homogeneous and heterogeneous assays, 250 ms delay time and 750 ms window time were used in order to achieve high signal intensities. The measurement temperature in homogeneous assays was 36 °C. 2.2. Conjugation of linker oligonucleotides to fab-fragments
Fig. 1. Principle of homogeneous one-step cTnI assay. Both labeled binders A and B consist of a Fab, a reporter oligonucleotide featuring one of the label moieties and a linker oligonucleotide, which is site-specifically conjugated to the Fab and connects the Fab to the label moiety. Reporter A is labeled with light-harvesting antenna ligand and reporter B is labeled with ion carrier chelate. The reporter oligonucleotides are designed to efficiently hybridize to corresponding linker oligonucleotides in the assay conditions, but they do not hybridize to each other and form a partial reporter stem when the analyte is not present, and thus the label moieties remain separate. When cTnI is present in the solution, Fabs bind simultaneously to it resulting in formation of the reporter stem and subsequent self-assembly of the luminescent mixed chelate complex.
The linker oligonucleotides A and B were covalently coupled from their maleimide group to Fabs 4C2 and MF4, respectively. The Fabs had been genetically engineered to contain a cysteine residue at the carboxyterminal end of the constant region of the heavy chain enabling site-specific coupling. Equivalent amounts (10 nmol) of Fab and oligonucleotide were used in conjugation reaction and the total reaction volume was 200 mL. The biomolecules were conjugated in 20 mM phosphate buffer, pH 7.4 at room temperature for 1.5 h and then transferred to 4 °C overnight. The Fab-linker oligonucleotide conjugates were purified with gel filtration using Superdex 75 h 10/30 column (Amersham Biosciences, Uppsala, Sweden). The column was equilibrated with 20 mM Tris buffer (pH 7.5) containing 300 mM NaCl and the same buffer was used for elution of the purified product. A flow of 0.2 mL/min was used for elution. The fractions containing the conjugate were pooled and concentrated using Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane (Merck Millipore Ltd., Cork, Ireland) and
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the buffer was changed to 20 mM Tris-buffer (pH 7.5) containing 150 mM NaCl and 10 μM ethylenediaminetetraacetic acid (EDTA). The oligonucleotide and protein concentrations from the pooled fractions were calculated based on absorbance measurements at 260 nm and 280 nm (Kukolka et al., 2004). 2.3. Conjugation of label moieties to reporter oligonucleotides Light-harvesting antenna ligand and europium(III) carrier chelate (Fig. S1) were synthesized and conjugated to terminal amino modifications of the reporter oligonucleotides as previously described (Karhunen et al., 2010, 2011; Päkkilä et al., 2013). Also the emission spectra and luminescence decay of the luminescent mixed chelate complex have been characterized earlier (Karhunen et al., 2010, 2011; Päkkilä et al., 2013). The reporters A and B included 15 nt complementary sequence with corresponding linker oligonucleotides and either 6, 7 or 8 nt long complementary stem sequence with the other reporter oligonucleotide. The labeled oligonucleotides were purified using a reverse-phase high-performance liquid chromatography (HPLC) with slight modifications to previously described protocol (Päkkilä et al., 2013). The reporter A6–8 oligonucleotides labeled with light-harvesting antenna ligand were purified using Hypersil ODS C18 column (150 mm 4.6 mm, 3 mm particle size) from Thermo Scientific (Waltham, MA) and a linear gradient from 14% to 30% acetonitrile in 50 mM aqueous triethylammonium acetate, pH 7.0 in 21 min. The reporter B6–8 oligonucleotides labeled with ion carrier chelate were purified using Syncronis C 18 column (5 mm particle size, 150 mm 4.6 mm) from Thermo Scientific and a linear gradient from 20% to 35% acetonitrile in 50 mM aqueous triethylammonium acetate, pH 7.0 in 20 min. The fractions containing the label conjugate were dried in MiVac Duo Concentrator (Genevac Inc., Stone Ridge, NY) and the dried conjugates were dissolved in 10 mM Tris buffer (pH 7.5), containing 50 mM NaCl and 10 mM EDTA. Labeling degree of the antenna ligand labeled oligonucleotide was calculated based on absorbance measurements at 260 nm and 330 nm to define oligonucleotide and antenna ligand concentrations, respectively. Extinction coefficient of 16,400 M 1 cm 1 was used for the antenna ligand concentration and oligonucleotide concentrations were calculated based on the assumption that OD 1 at 260 nm equals 33 mg/mL of ssDNA (Päkkilä et al., 2013). Eu(III) concentrations of the labeled fractions were determined using DELFIA technique (Wallac/PerkinElmer Life and Analytical Sciences) and a Eu(III) calibrator. 2.4. Optimization of the homogeneous assay The labeled binders were formed in the assay by adding equal amounts of reporter oligonucleotides and Fab-linker oligonucleotide conjugates to the reaction. To optimize the concentration of the labeled binders, three concentrations (1.3–5 nM in reaction) were studied. Also a small excess (1.5- or 2-fold) of antenna ligand-containing labeled binder A was studied to see if it would improve the assay performance without increasing the nonspecific background signal. Four cTnI concentrations in the range of 0–50 ng/mL in reaction were used in the experiments. Each concentration was pipetted in three replicates. The reagents were pipetted to wells to a total reaction volume of 60 mL and incubated for 30 min in slow shaking before time-resolved luminescence was measured. Different lengths (6–8 nt) of the complementary sequence of the reporter oligonucleotides forming the reporter stem were also evaluated. The length of the reporter stem was designed so that the melting temperature of the double strand was 11–22 °C according to DINAMelt Web Server (Markham and Zuker, 2005). Thus in the assay temperature of 36 °C, the reporter
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oligonucleotides did not hybridize to form the reporter stem without the analyte present. However, binding to the analyte increased the local concentration of the reporter oligonucleotides to enable the hybridization of the reporter stem and successive formation of luminescent lanthanide chelate complex. Six cTnIconcentrations in the range of 0–50 ng/mL, each in three replicates, were used in the assay and the concentration of each labeled binder was 2.5 nM in the reaction (60 mL). The wells were incubated in slow shaking in 36 °C for 30 min before the measurement of time-resolved luminescence. 2.5. Kinetics of the homogeneous assay The assay kinetics was measured at 36 °C using four cTnI concentrations in the range of 0–50 ng/mL in three replicates. The concentrations of the labeled binder A and B were 5 and 2.5 nM, respectively. The reagents were pre-heated for 10 min at 36 °C before the labeled binders and the analyte dilutions were combined and the reaction was started. The purpose of the preheating was to minimize the potential bias caused by the time required for the assay components to reach the assay temperature. The luminescence was followed for 33 min by measuring timeresolved luminescence using 12 s delay between measurements. 2.6. Standard curve of the homogeneous cTnI assay Fab-linker oligonucleotide conjugates and reporter oligonucleotides forming the labeled binders were pipetted to the microwell in a total volume of 30 mL. The final concentration of labeled binder A was 5 nM whereas the concentration of labeled binder B was 2.5 nM in the reaction. Twelve cTnI dilutions in the range of 0–250 ng/mL in reaction were pipetted to microwells in three replicates, except six for the blank. The total reaction volume was 60 mL and the reaction was incubated in 36 °C for 6 min before time-resolved luminescence was measured. A standard curve was formed from the data using nonlinear allometric fitting, and the LOD was calculated from the fitted curve based on 3 standard deviation of the blank. 2.7. Components of the heterogeneous reference assay To test the optimal configuration of the heterogeneous sandwich-type reference assay, both Fab 4C2 and Fab MF4 were labeled with intrinsically luminescent 9-dentate Eu(III)-chelate {2,2',2'',2'''-{[2-(4-Isothiocyanatophenyl)ethylimino]-bis(methylene) bis{4-{[4-(α-galactopyranoxy)phenyl]ethynyl}-pyridine-6,2-diyl]bis (methylenenitrilo)}tetrakis(acetato)}europium(III). The chelate was synthesized according to the protocol described earlier to an amino chelate intermediate (von Lode et al., 2003) and the iodoacetamido activation was introduced to the chelate (Takalo et al., 1994). The intrinsically luminescent 9-dentate Eu(III)-chelate was chosen for the reference assay because it had the same amount of coordination bonds shielding Eu(III) from quenching effect of the water molecules as the mixed chelate complex. Also the antenna chromophore was almost the same in both chelate structures although 9-dentate Eu(III)-chelate had two chromophores compared to one in the mixed chelate complex. For the labeling, 500 mg of Fab and 10-fold molar excess of 9-dentate Eu (III)-chelate were mixed in a reaction to a total reaction volume of 600 mL. The labeling buffer consisted of 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 8.5, 1 mM EDTA, 0.4 mM tris(2-carboxyethyl)phosphine. The reaction was incubated overnight at 4 °C, and the labeled antibody was purified from the excess label using fast performance liquid chromatography and Superdex 200 h 10/30 GL column (Amersham Biosciences). A flow of 15 mL/h and 50 mM Tris buffer (pH 7.75)
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containing 150 mM NaCl and 0.05% (w/v) NaN3 were used for the purification. The labeling degree of the purified product was calculated by dividing the Eu(III) concentration by the antibody concentration, which was determined by measuring the absorbance at 280 nm. The Eu(III) concentration was determined using DELFIA technique and Eu(III) calibrator. 2.8. Heterogeneous assay protocol Heterogeneous sandwich-type assay using the same Fabs MF4 and 4C2 was developed and used as a reference for evaluating the performance of the homogeneous assay. The same assay protocol was used both to study the optimal assay configuration and to create a standard curve. In the general assay protocol, the biotinylated Fab (50 ng) was pipetted into streptavidin-coated microtiter wells in 60 mL. The wells were incubated for 1 h at room temperature in slow shaking and then washed before pipetting the cTnI dilutions. For the optimization of assay configuration, four cTnI concentrations in the range of 0–100 ng/mL in volume of 60 mL were used, whereas thirteen concentrations in the range of 0–500 ng/mL were used for generating the standard curve. All standards were pipetted in three replicates except six for the blank. The wells were incubated for 1 h at 36 °C in 650 rpm shaking, and then washed twice. The Eu(III)-labeled Fab (50 ng) was added in 60 mL and incubated for 15 min at 36 °C in 650 rpm shaking before the wells were washed six times. The timeresolved luminescence was measured from dried wells.
3. Results and discussion 3.1. Components of the homogeneous assay Fab-linker oligonucleotide conjugate fractions were clearly separated from unconjugated linker oligonucleotide and Fab fractions in gel filtration. Reporter A6–8 oligonucleotides were labeled with light-harvesting antenna ligand. The antenna-labeled reporter was purified by HPLC and characterized with absorbance at 260 nm and 330 nm and the obtained labeling degrees were 1.4, 1.7 and 1.6 for reporter oligonucleotides having 6, 7 or 8 nt complementary region, respectively. The ion carrier chelate labeled reporter oligonucleotides B6, B7 and B8 resulted in labeling degrees of 1.0, 1.0 and 0.9, respectively. 3.2. Optimization of the homogeneous assay The choice of optimal concentration of the labeled binders consisting of Fab, linker oligonucleotide and reporter oligonucleotide was not straightforward. Lowering the concentrations of the labeled binders improved the signal-to-background ratios (Fig. 2). However, to be able to measure reliably analyte concentrations at the low end of the standard curve adequate signal levels compared to standard deviations are needed. Therefore labeled binder concentration of 2.5 nM instead of 1.3 nM was chosen for further experiments. This concentration resulted also in best LODs. Labeled binder concentrations above 5 nM were not evaluated because they were expected to have even lower signal-to-background ratios as observed in previous study (Päkkilä et al., 2013). The effect of labeled binder A excess on the assay performance was also studied. The twofold excess of the labeled binder A featuring the light-harvesting antenna ligand enhanced the signal levels slightly at low cTnI concentrations but it did not have significant effect on the signal-to-background ratios. Nevertheless, 5 nM labeled binder A was chosen for the assay to maximize the obtained signal levels at low cTnI concentrations.
Fig. 2. Optimization of the concentrations of the labeled binders in the homogeneous assay. The labeled binders consisted of Fab, linker oligonucleotide and reporter oligonucleotide that was coupled to a label moiety. The concentration of each labeled binder was 1.3 nM (squares), 2.5 nM (triangles) or 5 nM (diamonds) in the reaction. The equations of the allometric fittings were y¼ 323x0.958 (1.3 nM labeled binders), y¼649x0.896 (2.5 nM labeled binders) and y¼778x0.901 (5 nM labeled binders) and they resulted in LODs 0.15 ng/mL, 0.04 ng/mL and 0.05 ng/mL, respectively. Background signal of the zero calibrator has been subtracted from the signal intensities and the signal-to-background ratios are shown in the inset. The instrument background has been subtracted from the signals before calculating the signal-to-background ratio.
The reporter stem length was also optimized. The reporter B oligonucleotides had one nt overhang because it has earlier been described to enhance signal-to-background-ratios (Päkkilä et al., 2013). The best signal-to-background ratios (25 at 50 ng/mL cTnI) were obtained with reporter oligonucleotides forming a 6 nt long reporter stem (Fig. 3). With longer complementary sequences the signal levels were higher but the non-specific background increased even more implicating that the label moieties were brought together to some extent even without the analyte present. This caused lower signal-to-background ratios and thus the lowest cTnI concentrations could not be separated from the background as well as with the 6 nt reporter stem. Light-harvesting antenna ligand and ion carrier chelate have some affinity to each other, which contributes to the hybridization of the reporter stem. This may partly explain the high background signal observed with the long reporter stems although they should not form in the assay temperature according to the calculated Tm. The assay was performed at 36 °C, but lower assay temperature, such as room temperature, could be possible by further shortening the reporter stem. On the other hand, very short complementary sequences are not able to effectively bring the label moieties together, thus decreasing the obtained signal and impairing the assay performance. 3.3. Kinetics of the homogeneous assay The kinetics of the homogeneous assay was very fast as maximum signal-to-background ratios were obtained already after 6 min (Fig. 4). The signal intensity reached its maximum already after 3 min of incubation. However, the non-specific background was also high at the beginning of the assay because the assay components had time to cool during the pipetting thus enhancing the hybridization of the reporter stem and the formation of the
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Fig. 3. Optimization of the reporter stem length. Reporter oligonucleotides with 6 nt (squares), 7 nt (triangles) and 8 nt long reporter stem sequence (diamonds) were evaluated. The concentration of each labeled binder was 2.5 nM in the assay. The background signal of the zero calibrator was subtracted from the luminescence intensities. Signal-to-background ratios are shown in the inset. The instrument background has been subtracted from the signals before calculating the signal-tobackground ratio.
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Fig. 5. Standard curves of the homogeneous assay (squares) and the heterogeneous two-step assay (triangles). The intensity of the blank is subtracted from the data. LODs were 0.38 ng/mL (16 pM) for the homogeneous assay (dashed line) and 0.26 ng/mL (11.0 pM) for the heterogeneous reference assay (dotted line). The equations of the allometric fittings were y¼ 326x0.921, R2 ¼0.998 for homogeneous assay and y¼660x1.15, R2 ¼0.999 for heterogeneous assay.
3.4. Heterogeneous assay configuration Both Fabs were labeled and biotinylated in order to determine the optimal assay configuration. The labeling degree of Fabs MF4 and 4C2 were 2.3 and 0.7, respectively. When Eu(III)-Fab 4C2 was used for detection, the non-specific background was high resulting in low signal-to-background ratios, which were over forty times lower than with Eu(III)-Fab MF4 as tracer. Also the signal levels were significantly higher with Eu(III)-Fab MF4 and thus it was selected as tracer and biotinylated Fab 4C2 as capture for the heterogeneous assay (Fig. S3). 3.5. Assay performances of homogeneous assay and heterogeneous reference assay
Fig. 4. Kinetics of the homogeneous assay. The concentration of labeled binder A containing the antenna ligand was 5 nM and labeled binder B featuring the Eu(III) carrier chelate was 2.5 nM in the reaction. Kinetics was measured for cTnI concentrations of 7.5 ng/mL (squares), 20 ng/mL (triangles) and 50 ng/mL (diamonds).
mixed chelate complex. The background signal was decreased until 10 min and remained constant after that. After 15 min incubation the signal-to-background levels seemed to slowly decrease, which could be due to degradation of cTnI followed by decomposition of the mixed chelate complex (Fig. S2).
The LOD (defined as 3 SD of the blank) for homogeneous assay was 0.38 ng/mL (16 pM), which was better than in the previous study with FRET-based assay concept (Heyduk et al., 2008). More importantly, the LOD was comparable to that of the heterogeneous reference assay (0.26 ng/mL or 11 pM) (Fig. 5) even though it is known to be very difficult to reach the sensitivities of a heterogeneous assays with homogeneous assay format. This demonstrates the potential of switchable lanthanide luminescence for sensitive and rapid homogeneous protein detection. The assay buffer consisted 5 g/L bovine serum albumin and 0.5 g/L bovine γ-globulin, which are almost 5 million and over 250,000 times higher compared to the obtained LOD, respectively. The high abundance of other proteins did not interfere with the analyte recognition indicating high selectivity of the sensor. Despite the equal sensitivities obtained, differences between the homogeneous and the heterogeneous assays were also observed. Higher signal levels were obtained in the heterogeneous assay compared to the homogeneous assay because the 9-dentate Eu(III)-chelate in the heterogeneous assay had two chromophores. Therefore it was more luminous compared with the mixed chelate complex of the homogeneous assay, which had only one chromophore. The maximum signal-to-background ratio of the one-step
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homogeneous assay was 50 at cTnI-concentration of 250 ng/mL, which was very good for a homogeneous assay, but it did not reach the signal-to-background ratio of the heterogeneous two-step assay, which was 250 at the same standard point. The dynamic range of the homogeneous assay was over two orders of magnitude and it was limited by the hook-effect occurring at high analyte concentrations. In comparison, the dynamic range of the heterogeneous assay was over three orders of magnitude. CTnI is known to be very challenging biomarker because of the ultra-low sensitivities required for the assay. The LODs of both assays were approximately three orders of magnitude higher compared with recently published and commercially available high-sensitivity cardiac troponin assays, which have sensitivities at low ng/L range (Apple and Collinson, 2012; Apple et al., 2012; Hyytiä et al., 2013; Järvenpää et al., 2012). Thus the assays in their current form are not directly applicable for early detection of MI. Nevertheless, the obtained sensitivities would be adequate for many other analytes. Also, the obtained LODs were in the picomolar range such as the earlier protein detection assays using switchable lanthanide luminescence (Päkkilä et al., 2013; Karhunen et al., 2011). The commercial troponin assays have turn-around times around 15 min meeting the recommendation for assay times below 30 min for cardiac markers but only few novel cardiac troponin assays meet both requirements of speed and sensitivity although examples of either rapid or sensitive assays exist (Apple et al., 2004; Hyytiä et al., 2013; Song et al., 2011; Storrow et al. (2007); Zhang et al., 2011). The selectivity of an assay depends on the employed antibodies. Fabs 4C2 and MF4 have been used earlier as capture antibodies both individually and as a combination in a heterogeneous assay together with a third antibody, which was used for detection. In that assay, the use of serum samples did not cause any change in signal levels thus indicating high selectivity of the binders (Ylikotila et al., 2006). The homogeneous assay performance could be enhanced by further optimization of the complementary reporter stem sequences of the reporter oligonucleotides. The background signal can be lowered by using shorter complementary sequences of the reporter oligonucleotides, which would prevent the formation of the reporter stem at room temperature and subsequent lanthanide chelate self-assembly without the analyte present in the solution. The assay performance could also be improved by using more optimal binders for the assay. Fabs were required for this study in order to site-specifically conjugate oligonucleotides to binders. The Fabs 4C2 and MF4 used in this study had high affinities to cTnI (1.47 1010 and 2.2 1010 M 1, respectively) but they recognize the epitopes consisting of amino acids 23–29 and 190–196, respectively (Ylikotila et al., 2006). N- and C-terminal ends (amino acids 1–29 and 110–209) of cTnI are however known to be unstable regions due to susceptibility to proteolysis (Katrukha et al., 1998) and the problem is usually overcome by using antibodies recognizing the epitopes from the mid-fragment of the cTnI (Apple and Collinson, 2012; Panteghini, 2006). Due to limited availability of Fabs, we had to use Fabs recognizing epitopes from the terminal ends of cTnI. This is likely to affect the sensitivity of the assay and it also partially explains the difference in LODs compared with the high-sensitivity heterogeneous assays. Here, the same Fabs were used in both homogeneous and heterogeneous assays in order to avoid any bias to the assay performances caused by the binders.
4. Conclusions In this study, we demonstrated for the first time an antibody-based homogeneous assay using switchable lanthanide luminescence. By site-specific conjugation of oligonucleotide to Fab-fragment,
high-quality tailored reagents with exact positioning of the conjugation site and stoichiometric ratios of oligonucleotide and Fab were obtained. The study shows that switchable lanthanide luminescence effectively enables the development of homogeneous high performance rapid assays. The assay could be further simplified by using single maleimide modified oligonucleotide strands to directly connect label moieties to Fab-fragments. The applicability of switchable lanthanide luminescence for clinical samples is a subject for further studies.
Acknowledgments This work was funded by the Academy of Finland (Grant no. 132007). Jaana Rosenberg is thanked for the synthesis of lanthanide chelates and Pirjo Pietilä for producing the Fabs. Markus Vehniäinen is acknowledged for assistance in purification of Faboligonucleotide-conjugates.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.06.042.
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Rapid homogeneous immunoassay for cardiac troponin I using switchable lanthanide luminescence Henna Päkkilä n, Eeva Malmi, Satu Lahtinen, Tero Soukka Department of Biochemistry/Biotechnology, Tykistökatu 6A, 6th Floor, FI-20520 Turku, Finland
art ic l e i nf o
a b s t r a c t
Article history: Received 9 May 2014 Received in revised form 18 June 2014 Accepted 22 June 2014 Available online 25 June 2014
Homogeneous assays are advantageous because of their simplicity and rapid kinetics but typically their performance is severely compromised compared to heterogeneous assay formats. Here, we report a homogeneous immunoassay utilizing switchable lanthanide luminescence for detection and sitespecifically labeled recombinant antibody fragments as binders to improve the assay performance. Switchable lanthanide luminescence enabled elimination of assay background due to division of the luminescent lanthanide chelate into two non-luminescent label moieties. Simultaneous biomolecular recognition of model analyte cardiac troponin I by two antibody fragments brought the label moieties together and resulted in self-assembly of luminescent mixed chelate complex. The assay was very rapid as maximal signal-to-background ratios were achieved already after 6 min of incubation. Additionally, the limit of detection was 0.38 ng/mL (16 pM), which was comparable to the limit of detection for the heterogeneous reference assay based on the same binders (0.26 ng/mL or 11 pM). This is the first study to apply switchable lanthanide luminescence in immunoassays and demonstrates the versatile potential of the technology for rapid and sensitive homogeneous assays. & 2014 Elsevier B.V. All rights reserved.
Keywords: Lanthanide chelate Immunoassay Homogeneous detection Bioconjugation Cardiac troponin I
1. Introduction Homogeneous assay formats are well suited for point-of-care tests because they are simple to perform and have fast binding kinetics. However, matrix effects and high background signal are common drawbacks of the homogeneous assays. The challenge in using antibodies as binders in especially fluorescence-based homogeneous protein detection is their large size, which may take the labels too far apart in the formed immunocomplex to allow signal generation. Moreover, varying labeling degrees and random positions of the labels in the antibodies hamper the signal generation. The problems related to the use of antibodies in homogeneous assays have been avoided by using oligonucleotides attached to antibodies to enable nucleic acid amplification, such as in proximity-ligation assays (Darmanis et al., 2010; Fredriksson et al., 2002; Hammond et al., 2012) and binding-induced DNAassembly (Zhang et al., 2012). In these applications, the length of oligonucleotides can be adjusted depending on the size of an analyte to enable hybridization and following nucleic acid amplification. The use of direct fluorescence detection, such as Förster resonance energy transfer (FRET), avoids the need for timeconsuming nucleic acid amplification. Also in the FRET-based n
Corresponding author. Tel.: þ 358 2 333 8089. E-mail addresses: henna.pakkila@utu.fi (H. Päkkilä), esmalm@utu.fi (E. Malmi), selaht@utu.fi (S. Lahtinen), tejoso@utu.fi (T. Soukka). http://dx.doi.org/10.1016/j.bios.2014.06.042 0956-5663/& 2014 Elsevier B.V. All rights reserved.
assays, oligonucleotides have been used to assist in bringing the labels into close proximity to enable the generation of fluorescence signal (Heyduk et al., 2008, 2010). Lanthanide ions form brightly luminescent complexes when chelated to organic ligands containing light-harvesting chromophores. The ligand allows efficient excitation of the lanthanide ion and shields it from the quenching effects of water. The major advantage of lanthanide chelates is their long luminescence lifetime in millisecond range, which enables the use of timeresolved measurement to eliminate the short lifetime autofluorescence background. Furthermore, narrow emission bands and large Stokes shift are characteristic for lanthanide labels. Additional advantage of lanthanide chelate labels over conventional luminescent labels is that no self-quenching is observed enabling high labeling degree (Bünzli, 2010; Hemmilä et al., 1984). Switchable lanthanide luminescence is a novel label technology, which enables sensitive and specific homogeneous binding assays (Karhunen et al., 2010, 2011). The luminescent lanthanide chelate is split into two non-luminescent label moieties, which are covalently attached to two separate biomolecular binders. The binders recognize the analyte bringing the label moieties into close proximity, which increases the local concentration of the label moieties and enables lanthanide chelate self-assembly. Without the analyte, the label moieties remain apart and no luminescence signal is detected. The obtained signal is very specific to the
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presence of the analyte because two separate binding events are required for signal generation. The applicability of switchable lanthanide luminescence has been demonstrated in nucleic acid hybridization assays (Karhunen et al., 2010, 2013; Kitamura et al., 2008; Oser and Valet, 1990; Wang et al., 2001) and the principle has also been applied to nucleic acid amplification (Lehmusvuori et al., 2012, 2013) as well as to ligand and aptamer-based protein detection (Karhunen et al., 2011; Päkkilä et al., 2013). In this study, switchable lanthanide luminescence was used for the first time together with antibody binders. The label moieties were attached to fragment antigen binding portions of antibodies (Fabs) via oligonucleotides allowing direct detection and enabling rapid assay, which is more applicable for point-ofcare tests than those requiring nucleic acid amplification. The reporter oligonucleotides carrying the label moieties were hybridized to linker oligonucleotides that were covalently coupled to Fabs. The high background signal inherent to FRET-based assays and organic fluorophores was avoided by the use of long-lifetime labels and time-resolved luminescence measurement. The use of recombinant Fabs enabled site-specific coupling of linker oligonucleotides and prevented oligonucleotides from interfering with analyte recognition (Fig. 1). Replacing intact antibodies with Fabs has been shown to improve the detection limit of heterogeneous assays because more dense and well-oriented binding surface is obtained resulting in improved binding capacity and higher signal levels (Ylikotila et al., 2006). In addition, their use reduces the risk of interference caused by human anti-mouse antibodies in clinical samples (Hyytiä et al., 2013). Cardiac troponin I (cTnI) was used as a model analyte to demonstrate the potential of the assay concept. CTnI has high cardiac tissue specificity and clinical sensitivity, which makes it an ideal biomarker for the diagnosis of acute myocardial infarction (MI) (Thygesen et al., 2012). CTnI assays should have short turn-around times to enable early diagnosis and rapid treatment. While novel central laboratorybased tests are high-sensitivity assays, point-of-care tests with equal performance are still lacking (Lee-Lewandrowski et al., 2011).
Table 1 Oligonucleotide sequences. Name
Sequencea
Linker A Linker B Reporter Reporter Reporter Reporter Reporter Reporter
5′-#TTT TTT TTT TTT TTT TTT CCA GTT GCA GCA CGG-3′ 5′-GGA AGA CTC GAC AGC TTT TTT TTT TTT TTT TTT#-3′ 5′-*GCG TGG TTT TTC CGT GCT GCA ACT GG-3′ 5′-GCT GTC GAG TCT TCC TTT TTC CAC GCT*-3′ 5′-*GCG TGT GTT TTT CCG TGC TGC AAC TGG-3′ 5′-GCT GTC GAG TCT TCC TTT TTC ACA CGC T*-3′ 5′-*TGC GTG TGT TTT TCC GTG CTG CAA CTG G-3′ 5′-GCT GTC GAG TCT TCC TTT TTC ACA CGC AT*-3′
A6 B6 A7 B7 A8 B8
# Terminal maleimide modification, * terminal amino modification with six carbon aliphatic spacer. a Bolded nucleotides denote the reporter stem and underlined nucleotides the complementary region between linkers and reporters.
2. Materials and methods 2.1. Materials Human cardiac troponin complex was purchased from Hytest (Turku, Finland) and oligonucleotides (Table 1) were from Biomers. net (Ulm, Germany). Fab 4C2 and Fab MF4 used in homogeneous and heterogeneous assays were produced and biotinylated as previously described (Ylikotila et al., 2006). Assay buffer (50 mM Tris–HCl, pH 7.75 containing 150 mM NaCl, 0.05% (w/v) NaN3, 0.01% (w/v) Tween 40, 0.05% (w/v) bovine γ-globulin, 20 μM diethylenetriamine pentacetic acid and 0.5% (w/v) bovine serum albumin) and wash solution (5 mM Tris–HCl, pH 7.75 containing 150 mM NaCl, 0.005% (w/v) Tween 20 and 0.1% (w/v) Germall II) were from Kaivogen Oy (Turku, Finland) as well as the low fluorescence streptavidin-coated microplates used in heterogeneous reference assay. The low fluorescence 96-well MaxiSorp microtitration plates were from Nunc (Roskilde, Denmark). Victor Multilabel Plate Reader X4 used to measure the time-resolved luminescence was purchased from Wallac/Perkin-Elmer Life and Analytical Sciences (Wellesley, MA). Time-resolved Eu(III) luminescence intensities were measured using default factory-provided filters for 340 nm excitation and 615 nm emission. For the characterization of labeled assay components, both delay time and window time were 400 ms, but in the measurements of homogeneous and heterogeneous assays, 250 ms delay time and 750 ms window time were used in order to achieve high signal intensities. The measurement temperature in homogeneous assays was 36 °C. 2.2. Conjugation of linker oligonucleotides to fab-fragments
Fig. 1. Principle of homogeneous one-step cTnI assay. Both labeled binders A and B consist of a Fab, a reporter oligonucleotide featuring one of the label moieties and a linker oligonucleotide, which is site-specifically conjugated to the Fab and connects the Fab to the label moiety. Reporter A is labeled with light-harvesting antenna ligand and reporter B is labeled with ion carrier chelate. The reporter oligonucleotides are designed to efficiently hybridize to corresponding linker oligonucleotides in the assay conditions, but they do not hybridize to each other and form a partial reporter stem when the analyte is not present, and thus the label moieties remain separate. When cTnI is present in the solution, Fabs bind simultaneously to it resulting in formation of the reporter stem and subsequent self-assembly of the luminescent mixed chelate complex.
The linker oligonucleotides A and B were covalently coupled from their maleimide group to Fabs 4C2 and MF4, respectively. The Fabs had been genetically engineered to contain a cysteine residue at the carboxyterminal end of the constant region of the heavy chain enabling site-specific coupling. Equivalent amounts (10 nmol) of Fab and oligonucleotide were used in conjugation reaction and the total reaction volume was 200 mL. The biomolecules were conjugated in 20 mM phosphate buffer, pH 7.4 at room temperature for 1.5 h and then transferred to 4 °C overnight. The Fab-linker oligonucleotide conjugates were purified with gel filtration using Superdex 75 h 10/30 column (Amersham Biosciences, Uppsala, Sweden). The column was equilibrated with 20 mM Tris buffer (pH 7.5) containing 300 mM NaCl and the same buffer was used for elution of the purified product. A flow of 0.2 mL/min was used for elution. The fractions containing the conjugate were pooled and concentrated using Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane (Merck Millipore Ltd., Cork, Ireland) and
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the buffer was changed to 20 mM Tris-buffer (pH 7.5) containing 150 mM NaCl and 10 μM ethylenediaminetetraacetic acid (EDTA). The oligonucleotide and protein concentrations from the pooled fractions were calculated based on absorbance measurements at 260 nm and 280 nm (Kukolka et al., 2004). 2.3. Conjugation of label moieties to reporter oligonucleotides Light-harvesting antenna ligand and europium(III) carrier chelate (Fig. S1) were synthesized and conjugated to terminal amino modifications of the reporter oligonucleotides as previously described (Karhunen et al., 2010, 2011; Päkkilä et al., 2013). Also the emission spectra and luminescence decay of the luminescent mixed chelate complex have been characterized earlier (Karhunen et al., 2010, 2011; Päkkilä et al., 2013). The reporters A and B included 15 nt complementary sequence with corresponding linker oligonucleotides and either 6, 7 or 8 nt long complementary stem sequence with the other reporter oligonucleotide. The labeled oligonucleotides were purified using a reverse-phase high-performance liquid chromatography (HPLC) with slight modifications to previously described protocol (Päkkilä et al., 2013). The reporter A6–8 oligonucleotides labeled with light-harvesting antenna ligand were purified using Hypersil ODS C18 column (150 mm 4.6 mm, 3 mm particle size) from Thermo Scientific (Waltham, MA) and a linear gradient from 14% to 30% acetonitrile in 50 mM aqueous triethylammonium acetate, pH 7.0 in 21 min. The reporter B6–8 oligonucleotides labeled with ion carrier chelate were purified using Syncronis C 18 column (5 mm particle size, 150 mm 4.6 mm) from Thermo Scientific and a linear gradient from 20% to 35% acetonitrile in 50 mM aqueous triethylammonium acetate, pH 7.0 in 20 min. The fractions containing the label conjugate were dried in MiVac Duo Concentrator (Genevac Inc., Stone Ridge, NY) and the dried conjugates were dissolved in 10 mM Tris buffer (pH 7.5), containing 50 mM NaCl and 10 mM EDTA. Labeling degree of the antenna ligand labeled oligonucleotide was calculated based on absorbance measurements at 260 nm and 330 nm to define oligonucleotide and antenna ligand concentrations, respectively. Extinction coefficient of 16,400 M 1 cm 1 was used for the antenna ligand concentration and oligonucleotide concentrations were calculated based on the assumption that OD 1 at 260 nm equals 33 mg/mL of ssDNA (Päkkilä et al., 2013). Eu(III) concentrations of the labeled fractions were determined using DELFIA technique (Wallac/PerkinElmer Life and Analytical Sciences) and a Eu(III) calibrator. 2.4. Optimization of the homogeneous assay The labeled binders were formed in the assay by adding equal amounts of reporter oligonucleotides and Fab-linker oligonucleotide conjugates to the reaction. To optimize the concentration of the labeled binders, three concentrations (1.3–5 nM in reaction) were studied. Also a small excess (1.5- or 2-fold) of antenna ligand-containing labeled binder A was studied to see if it would improve the assay performance without increasing the nonspecific background signal. Four cTnI concentrations in the range of 0–50 ng/mL in reaction were used in the experiments. Each concentration was pipetted in three replicates. The reagents were pipetted to wells to a total reaction volume of 60 mL and incubated for 30 min in slow shaking before time-resolved luminescence was measured. Different lengths (6–8 nt) of the complementary sequence of the reporter oligonucleotides forming the reporter stem were also evaluated. The length of the reporter stem was designed so that the melting temperature of the double strand was 11–22 °C according to DINAMelt Web Server (Markham and Zuker, 2005). Thus in the assay temperature of 36 °C, the reporter
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oligonucleotides did not hybridize to form the reporter stem without the analyte present. However, binding to the analyte increased the local concentration of the reporter oligonucleotides to enable the hybridization of the reporter stem and successive formation of luminescent lanthanide chelate complex. Six cTnIconcentrations in the range of 0–50 ng/mL, each in three replicates, were used in the assay and the concentration of each labeled binder was 2.5 nM in the reaction (60 mL). The wells were incubated in slow shaking in 36 °C for 30 min before the measurement of time-resolved luminescence. 2.5. Kinetics of the homogeneous assay The assay kinetics was measured at 36 °C using four cTnI concentrations in the range of 0–50 ng/mL in three replicates. The concentrations of the labeled binder A and B were 5 and 2.5 nM, respectively. The reagents were pre-heated for 10 min at 36 °C before the labeled binders and the analyte dilutions were combined and the reaction was started. The purpose of the preheating was to minimize the potential bias caused by the time required for the assay components to reach the assay temperature. The luminescence was followed for 33 min by measuring timeresolved luminescence using 12 s delay between measurements. 2.6. Standard curve of the homogeneous cTnI assay Fab-linker oligonucleotide conjugates and reporter oligonucleotides forming the labeled binders were pipetted to the microwell in a total volume of 30 mL. The final concentration of labeled binder A was 5 nM whereas the concentration of labeled binder B was 2.5 nM in the reaction. Twelve cTnI dilutions in the range of 0–250 ng/mL in reaction were pipetted to microwells in three replicates, except six for the blank. The total reaction volume was 60 mL and the reaction was incubated in 36 °C for 6 min before time-resolved luminescence was measured. A standard curve was formed from the data using nonlinear allometric fitting, and the LOD was calculated from the fitted curve based on 3 standard deviation of the blank. 2.7. Components of the heterogeneous reference assay To test the optimal configuration of the heterogeneous sandwich-type reference assay, both Fab 4C2 and Fab MF4 were labeled with intrinsically luminescent 9-dentate Eu(III)-chelate {2,2',2'',2'''-{[2-(4-Isothiocyanatophenyl)ethylimino]-bis(methylene) bis{4-{[4-(α-galactopyranoxy)phenyl]ethynyl}-pyridine-6,2-diyl]bis (methylenenitrilo)}tetrakis(acetato)}europium(III). The chelate was synthesized according to the protocol described earlier to an amino chelate intermediate (von Lode et al., 2003) and the iodoacetamido activation was introduced to the chelate (Takalo et al., 1994). The intrinsically luminescent 9-dentate Eu(III)-chelate was chosen for the reference assay because it had the same amount of coordination bonds shielding Eu(III) from quenching effect of the water molecules as the mixed chelate complex. Also the antenna chromophore was almost the same in both chelate structures although 9-dentate Eu(III)-chelate had two chromophores compared to one in the mixed chelate complex. For the labeling, 500 mg of Fab and 10-fold molar excess of 9-dentate Eu (III)-chelate were mixed in a reaction to a total reaction volume of 600 mL. The labeling buffer consisted of 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 8.5, 1 mM EDTA, 0.4 mM tris(2-carboxyethyl)phosphine. The reaction was incubated overnight at 4 °C, and the labeled antibody was purified from the excess label using fast performance liquid chromatography and Superdex 200 h 10/30 GL column (Amersham Biosciences). A flow of 15 mL/h and 50 mM Tris buffer (pH 7.75)
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containing 150 mM NaCl and 0.05% (w/v) NaN3 were used for the purification. The labeling degree of the purified product was calculated by dividing the Eu(III) concentration by the antibody concentration, which was determined by measuring the absorbance at 280 nm. The Eu(III) concentration was determined using DELFIA technique and Eu(III) calibrator. 2.8. Heterogeneous assay protocol Heterogeneous sandwich-type assay using the same Fabs MF4 and 4C2 was developed and used as a reference for evaluating the performance of the homogeneous assay. The same assay protocol was used both to study the optimal assay configuration and to create a standard curve. In the general assay protocol, the biotinylated Fab (50 ng) was pipetted into streptavidin-coated microtiter wells in 60 mL. The wells were incubated for 1 h at room temperature in slow shaking and then washed before pipetting the cTnI dilutions. For the optimization of assay configuration, four cTnI concentrations in the range of 0–100 ng/mL in volume of 60 mL were used, whereas thirteen concentrations in the range of 0–500 ng/mL were used for generating the standard curve. All standards were pipetted in three replicates except six for the blank. The wells were incubated for 1 h at 36 °C in 650 rpm shaking, and then washed twice. The Eu(III)-labeled Fab (50 ng) was added in 60 mL and incubated for 15 min at 36 °C in 650 rpm shaking before the wells were washed six times. The timeresolved luminescence was measured from dried wells.
3. Results and discussion 3.1. Components of the homogeneous assay Fab-linker oligonucleotide conjugate fractions were clearly separated from unconjugated linker oligonucleotide and Fab fractions in gel filtration. Reporter A6–8 oligonucleotides were labeled with light-harvesting antenna ligand. The antenna-labeled reporter was purified by HPLC and characterized with absorbance at 260 nm and 330 nm and the obtained labeling degrees were 1.4, 1.7 and 1.6 for reporter oligonucleotides having 6, 7 or 8 nt complementary region, respectively. The ion carrier chelate labeled reporter oligonucleotides B6, B7 and B8 resulted in labeling degrees of 1.0, 1.0 and 0.9, respectively. 3.2. Optimization of the homogeneous assay The choice of optimal concentration of the labeled binders consisting of Fab, linker oligonucleotide and reporter oligonucleotide was not straightforward. Lowering the concentrations of the labeled binders improved the signal-to-background ratios (Fig. 2). However, to be able to measure reliably analyte concentrations at the low end of the standard curve adequate signal levels compared to standard deviations are needed. Therefore labeled binder concentration of 2.5 nM instead of 1.3 nM was chosen for further experiments. This concentration resulted also in best LODs. Labeled binder concentrations above 5 nM were not evaluated because they were expected to have even lower signal-to-background ratios as observed in previous study (Päkkilä et al., 2013). The effect of labeled binder A excess on the assay performance was also studied. The twofold excess of the labeled binder A featuring the light-harvesting antenna ligand enhanced the signal levels slightly at low cTnI concentrations but it did not have significant effect on the signal-to-background ratios. Nevertheless, 5 nM labeled binder A was chosen for the assay to maximize the obtained signal levels at low cTnI concentrations.
Fig. 2. Optimization of the concentrations of the labeled binders in the homogeneous assay. The labeled binders consisted of Fab, linker oligonucleotide and reporter oligonucleotide that was coupled to a label moiety. The concentration of each labeled binder was 1.3 nM (squares), 2.5 nM (triangles) or 5 nM (diamonds) in the reaction. The equations of the allometric fittings were y¼ 323x0.958 (1.3 nM labeled binders), y¼649x0.896 (2.5 nM labeled binders) and y¼778x0.901 (5 nM labeled binders) and they resulted in LODs 0.15 ng/mL, 0.04 ng/mL and 0.05 ng/mL, respectively. Background signal of the zero calibrator has been subtracted from the signal intensities and the signal-to-background ratios are shown in the inset. The instrument background has been subtracted from the signals before calculating the signal-to-background ratio.
The reporter stem length was also optimized. The reporter B oligonucleotides had one nt overhang because it has earlier been described to enhance signal-to-background-ratios (Päkkilä et al., 2013). The best signal-to-background ratios (25 at 50 ng/mL cTnI) were obtained with reporter oligonucleotides forming a 6 nt long reporter stem (Fig. 3). With longer complementary sequences the signal levels were higher but the non-specific background increased even more implicating that the label moieties were brought together to some extent even without the analyte present. This caused lower signal-to-background ratios and thus the lowest cTnI concentrations could not be separated from the background as well as with the 6 nt reporter stem. Light-harvesting antenna ligand and ion carrier chelate have some affinity to each other, which contributes to the hybridization of the reporter stem. This may partly explain the high background signal observed with the long reporter stems although they should not form in the assay temperature according to the calculated Tm. The assay was performed at 36 °C, but lower assay temperature, such as room temperature, could be possible by further shortening the reporter stem. On the other hand, very short complementary sequences are not able to effectively bring the label moieties together, thus decreasing the obtained signal and impairing the assay performance. 3.3. Kinetics of the homogeneous assay The kinetics of the homogeneous assay was very fast as maximum signal-to-background ratios were obtained already after 6 min (Fig. 4). The signal intensity reached its maximum already after 3 min of incubation. However, the non-specific background was also high at the beginning of the assay because the assay components had time to cool during the pipetting thus enhancing the hybridization of the reporter stem and the formation of the
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Fig. 3. Optimization of the reporter stem length. Reporter oligonucleotides with 6 nt (squares), 7 nt (triangles) and 8 nt long reporter stem sequence (diamonds) were evaluated. The concentration of each labeled binder was 2.5 nM in the assay. The background signal of the zero calibrator was subtracted from the luminescence intensities. Signal-to-background ratios are shown in the inset. The instrument background has been subtracted from the signals before calculating the signal-tobackground ratio.
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Fig. 5. Standard curves of the homogeneous assay (squares) and the heterogeneous two-step assay (triangles). The intensity of the blank is subtracted from the data. LODs were 0.38 ng/mL (16 pM) for the homogeneous assay (dashed line) and 0.26 ng/mL (11.0 pM) for the heterogeneous reference assay (dotted line). The equations of the allometric fittings were y¼ 326x0.921, R2 ¼0.998 for homogeneous assay and y¼660x1.15, R2 ¼0.999 for heterogeneous assay.
3.4. Heterogeneous assay configuration Both Fabs were labeled and biotinylated in order to determine the optimal assay configuration. The labeling degree of Fabs MF4 and 4C2 were 2.3 and 0.7, respectively. When Eu(III)-Fab 4C2 was used for detection, the non-specific background was high resulting in low signal-to-background ratios, which were over forty times lower than with Eu(III)-Fab MF4 as tracer. Also the signal levels were significantly higher with Eu(III)-Fab MF4 and thus it was selected as tracer and biotinylated Fab 4C2 as capture for the heterogeneous assay (Fig. S3). 3.5. Assay performances of homogeneous assay and heterogeneous reference assay
Fig. 4. Kinetics of the homogeneous assay. The concentration of labeled binder A containing the antenna ligand was 5 nM and labeled binder B featuring the Eu(III) carrier chelate was 2.5 nM in the reaction. Kinetics was measured for cTnI concentrations of 7.5 ng/mL (squares), 20 ng/mL (triangles) and 50 ng/mL (diamonds).
mixed chelate complex. The background signal was decreased until 10 min and remained constant after that. After 15 min incubation the signal-to-background levels seemed to slowly decrease, which could be due to degradation of cTnI followed by decomposition of the mixed chelate complex (Fig. S2).
The LOD (defined as 3 SD of the blank) for homogeneous assay was 0.38 ng/mL (16 pM), which was better than in the previous study with FRET-based assay concept (Heyduk et al., 2008). More importantly, the LOD was comparable to that of the heterogeneous reference assay (0.26 ng/mL or 11 pM) (Fig. 5) even though it is known to be very difficult to reach the sensitivities of a heterogeneous assays with homogeneous assay format. This demonstrates the potential of switchable lanthanide luminescence for sensitive and rapid homogeneous protein detection. The assay buffer consisted 5 g/L bovine serum albumin and 0.5 g/L bovine γ-globulin, which are almost 5 million and over 250,000 times higher compared to the obtained LOD, respectively. The high abundance of other proteins did not interfere with the analyte recognition indicating high selectivity of the sensor. Despite the equal sensitivities obtained, differences between the homogeneous and the heterogeneous assays were also observed. Higher signal levels were obtained in the heterogeneous assay compared to the homogeneous assay because the 9-dentate Eu(III)-chelate in the heterogeneous assay had two chromophores. Therefore it was more luminous compared with the mixed chelate complex of the homogeneous assay, which had only one chromophore. The maximum signal-to-background ratio of the one-step
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homogeneous assay was 50 at cTnI-concentration of 250 ng/mL, which was very good for a homogeneous assay, but it did not reach the signal-to-background ratio of the heterogeneous two-step assay, which was 250 at the same standard point. The dynamic range of the homogeneous assay was over two orders of magnitude and it was limited by the hook-effect occurring at high analyte concentrations. In comparison, the dynamic range of the heterogeneous assay was over three orders of magnitude. CTnI is known to be very challenging biomarker because of the ultra-low sensitivities required for the assay. The LODs of both assays were approximately three orders of magnitude higher compared with recently published and commercially available high-sensitivity cardiac troponin assays, which have sensitivities at low ng/L range (Apple and Collinson, 2012; Apple et al., 2012; Hyytiä et al., 2013; Järvenpää et al., 2012). Thus the assays in their current form are not directly applicable for early detection of MI. Nevertheless, the obtained sensitivities would be adequate for many other analytes. Also, the obtained LODs were in the picomolar range such as the earlier protein detection assays using switchable lanthanide luminescence (Päkkilä et al., 2013; Karhunen et al., 2011). The commercial troponin assays have turn-around times around 15 min meeting the recommendation for assay times below 30 min for cardiac markers but only few novel cardiac troponin assays meet both requirements of speed and sensitivity although examples of either rapid or sensitive assays exist (Apple et al., 2004; Hyytiä et al., 2013; Song et al., 2011; Storrow et al. (2007); Zhang et al., 2011). The selectivity of an assay depends on the employed antibodies. Fabs 4C2 and MF4 have been used earlier as capture antibodies both individually and as a combination in a heterogeneous assay together with a third antibody, which was used for detection. In that assay, the use of serum samples did not cause any change in signal levels thus indicating high selectivity of the binders (Ylikotila et al., 2006). The homogeneous assay performance could be enhanced by further optimization of the complementary reporter stem sequences of the reporter oligonucleotides. The background signal can be lowered by using shorter complementary sequences of the reporter oligonucleotides, which would prevent the formation of the reporter stem at room temperature and subsequent lanthanide chelate self-assembly without the analyte present in the solution. The assay performance could also be improved by using more optimal binders for the assay. Fabs were required for this study in order to site-specifically conjugate oligonucleotides to binders. The Fabs 4C2 and MF4 used in this study had high affinities to cTnI (1.47 1010 and 2.2 1010 M 1, respectively) but they recognize the epitopes consisting of amino acids 23–29 and 190–196, respectively (Ylikotila et al., 2006). N- and C-terminal ends (amino acids 1–29 and 110–209) of cTnI are however known to be unstable regions due to susceptibility to proteolysis (Katrukha et al., 1998) and the problem is usually overcome by using antibodies recognizing the epitopes from the mid-fragment of the cTnI (Apple and Collinson, 2012; Panteghini, 2006). Due to limited availability of Fabs, we had to use Fabs recognizing epitopes from the terminal ends of cTnI. This is likely to affect the sensitivity of the assay and it also partially explains the difference in LODs compared with the high-sensitivity heterogeneous assays. Here, the same Fabs were used in both homogeneous and heterogeneous assays in order to avoid any bias to the assay performances caused by the binders.
4. Conclusions In this study, we demonstrated for the first time an antibody-based homogeneous assay using switchable lanthanide luminescence. By site-specific conjugation of oligonucleotide to Fab-fragment,
high-quality tailored reagents with exact positioning of the conjugation site and stoichiometric ratios of oligonucleotide and Fab were obtained. The study shows that switchable lanthanide luminescence effectively enables the development of homogeneous high performance rapid assays. The assay could be further simplified by using single maleimide modified oligonucleotide strands to directly connect label moieties to Fab-fragments. The applicability of switchable lanthanide luminescence for clinical samples is a subject for further studies.
Acknowledgments This work was funded by the Academy of Finland (Grant no. 132007). Jaana Rosenberg is thanked for the synthesis of lanthanide chelates and Pirjo Pietilä for producing the Fabs. Markus Vehniäinen is acknowledged for assistance in purification of Faboligonucleotide-conjugates.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.06.042.
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