High-Sensitivity Immunoassay with Surface Plasmon Field-Enhanced

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High-Sensitivity Immunoassay with Surface Plasmon Field-Enhanced Fluorescence Spectroscopy Using a Plastic Sensor Chip: Application to Quantitative Analysis of Total Prostate-Specific Antigen and GalNAcβ1−4GlcNAc-Linked Prostate-Specific Antigen for Prostate Cancer Diagnosis Takatoshi Kaya,*,† Tomonori Kaneko,† Shun Kojima,† Yukito Nakamura,† Youichi Ide,† Kenji Ishida,† Yoshihiko Suda,† and Katsuko Yamashita‡ †

Corporate R&D Headquarters, Konica Minolta, Inc., No. 1 Sakura-machi, Hino-shi, Tokyo 191-8511, Japan Department of Histology and Cell Biology, School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0027, Japan

‡

ABSTRACT: A high-sensitivity immunoassay system with surface plasmon field-enhanced fluorescence spectrometry (SPFS) was constructed using a plastic sensor chip and then applied to the detection of total prostate-specific antigen (total PSA) and GalNAcβ1−4GlcNAc-linked prostate-specific antigen (LacdiNAcPSA) in serum, to discriminate between prostate cancer (PC) and benign prostate hyperplasia (BPH). By using this automated SPFS immunoassay, the detection limit for total PSA in serum was as low as 0.04 pg/mL, and the dynamic range was estimated to be at least five digits. A two-step sandwich SPFS immunoassay for LacdiNAcPSA was constructed using both the anti-PSA IgG antibody to capture PSA and Wisteria f loribunda agglutinin (WFA) for the detection of LacdiNAc. The results of the LacdiNAc-PSA immunoassay with SPFS showed that the assay had a sensitivity of 20.0 pg/mL and permitted the specific distinction between PC and BPH within the PSA gray zone. These results suggested that highsensitivity automated SPFS immunoassay systems might become a powerful tool for the diagnosis of PC and other diseases.

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diagnosis technology. The second problem with CLEIA is that the enzymatic reaction requires accurate control of temperature in order to guarantee assay reproducibility.3 This requirement has resulted in not only an increase in the size of the assay machine to ensure thermal capacity but also limitation of the reaction temperature to the range of 35−42 °C for an optimal enzymatic reaction. Thus, overcoming these limitations may allow for the development of novel diagnostic assays in the medical field. The application of new biotechnologies, biotechnology-based drugs, and tissue engineering applications are expected to permit quantitative determination of glycan structures (particularly those expressed specifically at the cell membrane interface), certain serum glycoproteins found in various diseases, and functional roles of glycoproteins.6,7 Although the glycan structures in purified glycoconjugates have typically been identified using nuclear magnetic resonance spectrometry,8 matrix-assisted laser desorption/ionization-time-of-flightmass spectrometry,9 and methylation analysis in combination with exoglycosidase digestion,10 these methods are difficult to

dvancements in molecular cell biology in the past decade have enabled the development of various cutting-edge biotechnologies using biotechnology-based drugs and tissue engineering.1,2 These advanced biotechnologies or biomaterials require a high level of quality control for successful clinical investigation and commercialization. Thus, superior evaluation technology is essential for high-quality, multifaceted, and precise management of novel biotechnologies. Chemiluminescent enzyme immunoassays (CLEIAs), used for the quantitative measurement of biomarkers in serum or plasma, occupy a central position in medical diagnostic technology.3 CLEIA uses magnetic particle technology and an enzyme label to achieve both high reaction efficiency and amplification of the immunoassay signal. Many marketed, highsensitivity immunoassay systems have adopted the CLEIA principle, and this assay format now represents one of the primary diagnostic systems used in clinical applications worldwide. However, there is room for improvement, particularly in at least two CLEIA components. The first is that the chemiluminescent reaction has an extremely low efficiency in the aqueous phase.4 According to a study on the bioluminescent enzyme immunoassay (BLEIA), this technology has a much more efficient luminescent reaction than that of CLEIA,5 demonstrating a technical paradigm shift in the field of © 2014 American Chemical Society

Received: October 6, 2014 Accepted: December 29, 2014 Published: December 29, 2014 1797

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Analytical Chemistry

efficiency expected for clinically relevant samples in both steps. Therefore, in the field of glycoprotein analysis, a solution is needed that allows both flexibility for assay conditions and sensitivity for the detection of target molecules. Here, we demonstrate the development and evaluation of an automated SPFS immunoassay system with a disposable plastic sensor chip and compare it with ELISA under the same material conditions as for total PSA immunoassays. We also developed a LacdiNAc-PSA immunoassay that uses an automated SPFS lectin immunoassay system employing βGalNAc-specific Wisteria f loribunda agglutinin (WFA), with a fluorescent label. On the basis of this established methodology, we investigated whether the LacdiNAc-PSA immunoassay could be used to distinguish between PC and BPH within the total PSA gray zone (4−20 ng/mL total PSA) using the SPFS system.

apply to small quantities and low-purity samples. On the other hand, some lectin-based methods, including lectin column chromatography,11 lectin affinity electrophoresis,12 lectin microarrays,13 and lectin immunoassays,14 have been used to determine glycan motifs in glycoproteins without sample restriction. However, these lectin-based methods have long assay times, require complicated procedures, are not quantitative, and have low sensitivity, all of which are distinct disadvantages. Since the quantitative analysis of binding between lectin and glycoprotein can be efficiently performed at a low temperature,15 an automated CLEIA system,3 which maintains a high temperature during an enzymatic reaction, presents theoretical difficulties in the high-sensitivity detection or determination of glycan structures using lectins. Surface plasmon-field enhanced fluorescence spectroscopy (SPFS) was developed in earnest by several groups and is expected to be a technology with one of the highest detection sensitivities that does not use an enzyme reaction for amplification.16−18 As a model assay for this system, a free prostate-specific antigen (free PSA) immunoassay using SPFS was carried out at room temperature.16 This result suggested that SPFS could be developed into a highly sensitive, simple immunoassay system that does not require an enzyme reaction and can be carried out at room temperature. However, one of the reasons that SPFS has not become a more general analytical method (e.g., as compared with surface plasmon resonance [SPR])19 is that SPFS requires advanced optical techniques and comprehensive design techniques for the sensor surface and fluorescent label to achieve highly reproducible measurements.16−18 Thus, development of a disposable SPFS sensor chip may be an effective solution to overcome these problems and facilitate the clinical use of this technology. PSA is frequently employed as the model material for evaluation of immunoassay systems or measurements5,20−22 and has previously been used to assess the validity of the SPFS system.16 PSA is an intercellular glycoprotein (34 kDa kallikrein-like protease) that is locally synthesized in prostatic tissue, and serum PSA exists as both free PSA and the antichymotrypsin (ACT)-PSA complex (ACT-PSA), which are collectively referred to as total PSA.23,24 Although total PSA is a useful diagnostic marker of prostate cancer (PC),25 the low specificity of total PSA in distinguishing between PC and benign prostate hyperplasia (BPH) will need to be addressed in order to improve the overtreatment of PC.26 Pro-PSA,27 the 4kpanel,28 and free PSA29 were recently developed as new biomarkers used to discriminate PC from BPH. We have recently reported that changes in the N-glycan structures of PSA, which allow for differentiation between PC and BPH, can be detected using a Trichosanthes japonica agglutinin-II (TJA-II)-Sepharose column chromatography combined with an ELISA method.30 This method completely satisfies the sensitivity and specificity requirements for quantitative analysis of GalNAcβ1−4GlcNAc-linked prostatespecific antigen (LacdiNAc-PSA) in the serum of patients with PC and BPH; however, a sample concentration step is required before the ELISA procedure, and the total assay takes several hours.30 On the other hand, the lectin sandwich immunoassay is considered a relatively easy and widely acceptable method in the field of glycoprotein analysis.31 Lectin sandwich immunoassays can be subdivided into two steps: lectin capture and lectin detection. However, the presence of carbohydrate moieties in many serum glycoprotein molecules often results in far worse sensitivity and far lower specificity than the

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MATERIALS AND METHODS Materials. PSA purified from human seminal fluid was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). The standard total PSA reagent was prepared by dilution of PSA antigen material with PSA-negative female serum. Antitotal PSA monoclonal antibodies (clones PS2 and PS6) were purchased from HyTest Ltd. (Turku, Finland), and No. 72 was purchased from Mikuri Immunological Laboratories Co., Ltd., (Osaka, Japan). WFA, WFA agarose, and Vicia villosa lectin (VVL) were purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). Soybean agglutinin (SBA) was purchased from J-Oil Mills, Inc. (Tokyo, Japan). TJA-II was purified according to previously described methods.32 NHydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). LNCaP (RCB2144), a human PC cell line, was provided by the European collection of Animal Cell Cultures Catalogue (ECACC) (Porton Down, UK). LNCaP cells were cultured in RPMI 1640 medium (Life Technologies, Inc., Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS) at 37 °C in an incubator with an atmosphere of 5% CO2. The cell culture supernatant without any treatment was used as a LacdiNAcPSA control for SPFS measurements. Sialidase-treated human urinary Tamm-Horsfall glycoprotein (THGP, SciPac Ltd., Sittingbourne, Kent, UK) was used for screening β-GalNAc-specific lectins that could have applications in high-sensitivity lectin immunoassays. Since THGP possesses a GalNAcβ1 → 4(NeuAcα2 → 3)Galβ1 → 4GlcNAc moiety (Sd blood group antigenic determinant) in its N-glycans,33 it was digested with Arthrobacter sialidase (50 milliunits of enzyme at pH 4.5, in 0.1 M acetate buffer for 2 h) to obtain THGP-exposed β-GalNAc residues at the nonreducing terminal. Sera. The sera used in this study were purchased from ProteoGenex Inc. (CA, USA), ProMedoDx LLC. (MA, USA), Asterand Inc. (Detroit, MI, USA), and BioServe Biotechnologies, Ltd. (MD, USA). The sera were randomly selected from 44 patients who were diagnosed with PC at various clinical stages and from 27 patients with BPH. Affinity Analysis of Anti-PSA IgG to PSA and βGalNAc-Specific Lectins to THGP by SPR. The binding affinities for antitotal PSA IgG to total PSA and for several βGalNAc-specific lectins to sialidase-treated THGP were measured by SPR assays using a BIAcore 2000 instrument. Antitotal PSA monoclonal antibodies (PS2, PS6, and No. 72) 1798

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Analytical Chemistry were immobilized on a CM5 sensor chip (GE Healthcare BioScience AB, Uppsala, Sweden) by the amide-coupling method using 100 mM NHS and 100 mM EDC. The coupling densities of all antibodies were controlled at 2000 RU. Total PSA was diluted in HPS-EP (0.01 M HEPES-NaOH [pH 7.4], 150 mM NaCl, 0.005% [v/v] polysorbate20 [GE Healthcare Bio-Science AB]) buffer and injected onto the sensor chip at 5 μL/min. Various concentrations of total PSA were introduced onto the anti-PSA monoclonal antibody-captured surfaces. SPR affinity analysis of β-GalNAc-specific lectins, including TJA-II, VVL, SBA, and WFA, was carried out using sialidasetreated THGP as a model glycoprotein that possesses βGalNAc residues at its nonreducing terminal. Sialidase-treated THGP was immobilized on a CM5 sensor chip by the amidecoupling method using 100 mM NHS and 100 mM EDC. The coupling density of THGP was 3500 RU. The lectins were diluted in HPS-P (0.01 M HEPES-NaOH [pH 7.4], 150 mM NaCl [GE Healthcare Bio-Science AB]) buffer, and various concentrations of each lectin were introduced onto the THGPcaptured surface for 180 s at a flow rate of 30 μL/min. The entire interaction assay was carried out at 25 °C, subtracting the surface signals with pure buffer via calculation of the dissociation constants using the 1:1 Langmuir binding mode and BIA evaluation 3.0 software (GE Healthcare Bio-Science AB). Total PSA ELISA. ELISA was used as a comparative method, the sole difference being that the detection antibody was PS6. Antibody PS2 was coated in 96-well plates (Maxi Sorp F96, Thermo Fisher Scientific Inc., MA, USA) at 0.5 μg/well at pH 9.6 in carbonate buffer and incubated overnight at 4 °C. The wells were washed with PBS-T and blocked for 2 h at room temperature with 200 μL of 1% bovine serum albumin (BSA) in PBS. Next, 100 μL of total PSA diluted in PSAnegative female serum (R161824, Kohjin Bio Co., Ltd., Saitama, Japan) was added to each well. Plates were incubated at room temperature for 1 h and then washed three times with PBS-T. Biotinylated PS6 antibodies were prepared using a Biotin-Labeling Kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and diluted with 1% BSA-PBS. Next, 0.1 μg was added to each well, and plates were incubated for 1 h at room temperature. After the plates were washed with PBS-T, 0.0125 μg/mL streptavidin-conjugated horseradish peroxidase (Thermo Fisher Scientific Inc. [Pierce]) in 1% BSA-PBS was added, and plates were incubated for an additional 30 min. After several additional washes with PBS-T, Super Signal ELISA Femto Substrate (Thermo Fisher Scientific Inc.) was added as the chemiluminescence substrate for 5 min at room temperature, and luminescence signals were quantified by a microplate reader (SH-9000; Corona Electric Co., Ltd., Ibaraki, Japan). Preparation of the SPFS Disposable Plastic Sensor Chip. We fabricated the SPFS disposable plastic sensor chip, which was prepared in-house using an injection molding technique, as shown in Figure 1. We first fabricated a selfassembled monolayer (SAM) using amine-terminated thiol (11-amino-1-undecanethiol, hydrochloride, Dojindo Molecular Technologies, Inc.) on a gold membrane, which was prepared by magnetron sputtering (L-430S-FHS, Canon Anelva Corp., Kanagawa, Japan) with a thickness of 50 nm on the upper part of the base of the trapezoidal plastic molding prism. Carboxymethyl dextran (CMD; Meito Sangyo Co., Ltd., Nagoya, Japan) and anti-PSA IgG were then sequentially immobilized by an amide coupling reaction (100 mM NHS and 100 mM EDC) on the gold membrane through the SAM and

Figure 1. Schematic view of the automatic SPFS immunoassay system with a disposable plastic sensor chip.

CMD molecules. Finally, a cover plate that included a mixing chamber was bonded by black double-sided adhesion tape to a prism after completion of the blocking step using 1% BSA-PBS (Blocker BSA in 10× PBS; Pierce) following the anti-PSA antibody immobilization. The bonding process created a microchannel (100 μm high, 3 mm wide, and 30 mm long) between the cover plate and the trapezoidal prism (Figure 1). Two-Step Sandwich SPFS Immunoassay with Disposable Sensor Chip. Two-step sandwich SPFS immunoassays of total PSA and LacdiNAc-PSA were carried out automatically by moving a cylindrical pump between the disposable sensor chip and a reagent container in a self-developed assay machine (Figure 1). The reagent container already contained a number of separate reagents, including wash buffer (TBS-0.05% Tween20, 10× TBS [Nippon Gene Co., Ltd., Tokyo, Japan] and polysorbate 20 [MP Biomedicals, LLC., CA, USA]), AF647PS6 antibody, or AF647-WFA (prepared using an Alexa Fluor 647 labeling kit [A20186, Thermo Fisher Scientific Inc.]) and the measurement sample. In the total PSA immunoassay and the LacdiNAc-PSA immunoassay, the 100 μL measurement sample and AF647 labeled with either antibody or the lectin solution were allowed to react for 30 and 10 min, respectively, and excess antibody or lectin solution was removed by washing with buffer after each reaction. The concentration of the AF647-labeled antibody and the lectin solution was set to 1.0 μg/mL using 1% BSA-PBS. After washing four times with PBST, the final washing buffer was left in the disposable sensor chip for SPFS measurement. All assays were carried out automatically at 25 °C for 45 min; four immunoassays were carried out simultaneously. Optical Construction of an SPFS Instrument. Optical construction of the SPFS instrument is described schematically in Figure 2. The fluorescent signal that passed through the emission filter (DIF-BP-1 [half width: 668 ± 5 nm], Optical Coatings Japan, Tokyo, Japan) was detected by a photomultiplier tube (H7421-40, Hamamatsu Photonics K.K., Shizuoka, Japan), which was located at the end of a lightconverging optical system (NA = 0.6; Edmund Optics Japan Ltd., Japan, Tokyo). The SPFS system allowed simultaneous detection of reflectivity as well as fluorescence intensity as a function of incidence angle or time. The reflective intensity was measured by a photodiode (S2281-04, Hamamatsu Photonics K.K.). A laser diode (635 nm, 0.95 mW; Edmund Optics Japan Ltd.) was used as a light source with a Neutral Density filter (AND20C-10 [10%], Sigmakoki Co., Ltd., Saitama, Japan). The laser light was already p-polarized and collimated by the internal laser diode system. Statistical Analysis. All statistical analyses were performed using Stat Flex 6.0 software (Artech Co., Ltd., Osaka, Japan; 1799

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Figure 2. Schematic view of the optical construction of the SPFS instrument.

URL: http://www.statflex.net/). The differences between PC and BPH in median levels of total PSA and LacdiNAc-PSA were examined using the Mann−Whitney U test with two-sided p values.

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RESULTS AND DISCUSSION Construction of SPFS Immunoassay System with Disposable Sensor Chip. In order to establish highersensitivity lectin immunoassay methods, we first created an SPFS immunoassay system that used a disposable sensor chip. SPFS, one of the most sensitive fluorescence detection technologies, is based on a plasmon-enhanced fluorescent signal and has an extremely low background signal.16−18 SPFS utilizes an enhanced electric field to excite the fluorescent probe located close to the metal/water interface. However, SPFS is problematic in that locating fluorescent molecules adjacent to a metal surface causes extinction by energy transfer to the metal. To solve this problem, Knoll and co-workers17 used a threedimensionally extended surface matrix, displacing the interaction platform away from the metal surface; they also selected AF647 as a quenchless fluorescent probe for quantitative measurement.17 In this study, we further developed the automatic SPFS immunoassay system using a disposable plastic sensor chip, which was prepared in-house using an injection molding technique, as shown in Figure 1. All of the immunoassay reagent solutions were transported in the microchannel between a disposable dispenser tip and the mixing chamber using a cylindrical pump (Figure 1). The mixing chamber, which was designed using fluidics technology simulation, effectively supported the efficiency of the immunoassay reaction by reducing the heterogeneous concentration gradient caused by immunoreaction of total PSA and LacdiNAc-PSA. This high-efficiency reaction technology, combined with a high-density antibody surface, improved the sensitivity of the SPFS immunoassay system under a limited amount of sample. Evaluation of the Disposable Sensor Chip by SPR/ SPFS Measurements. The reflection intensity curves (Figure 3a) and the fluorescence intensity curves (Figure 3b) were acquired by scanning (scan rate: 0.05 deg/step) the incidence angle under the immunoassay, which was carried out with different total PSA concentrations (0.0512−32.0 pg/mL) in each disposable sensor chip. The reflection intensity curves (Figure 3a) clearly showed that the minimum reflection intensity associated with the SPR angle was at about 72°, and

Figure 3. Reflection intensity curves (a) and fluorescent intensity curves (b) acquired by scanning the incidence angles at different concentrations (0.0512−32 pg/mL) of total PSA in the immunoassay for each plastic sensor chip.

the maximum recorded intensity was at a total reflective angle of about 63°. Notably, the stability of both the SPR angles and the total reflection angles in each disposable chip showed not only the high reproducibility of the sensor’s optical properties achieved by injection molding fabrication but also the low sensitivity of SPR measurement within a low range of antigen concentrations. Conversely, on examination of the results of SPFS measurement (Figure 3b), the maximum fluorescence intensities of curves clearly depended on the total PSA concentrations and were recorded at a nearly identical incidence angle. Additionally, the system noise of SPFS measurement (Figure 3b), which was measured before using the AF-647 labeled antibody reaction in this study, was very stable, with a value of about 1100 counts per seconds (cps). On the basis of these results, we chose an incidence angle of 71° and deducted the system noise signal of the SPFS immunoassay system from all of the SPFS fluorescent intensity data. Comparison of Total PSA Sensitivities for the SPFS Immunoassay and ELISA. The total PSA calibration curves of the SPFS immunoassay and ELISA are shown in Figure 4. The results of these calibration curves showed that the detection limits (LODs: means of six replicates of the zero standard plus three standard deviations [SDs]) of the SPFS immunoassay and ELISA were 0.04 and 20.0 pg/mL total PSA in 100 μL serum, respectively, suggesting that the SPFS immunoassay was at least 500 times more sensitive than ELISA under the same material conditions. The limit of quantitation (LOQ: coefficient of variation [CV] 10% or less of six replicates) of the SPFS immunoassay was 0.256 pg/mL the total PSA. This result demonstrates the most sensitive SPFS immunoassay to date, with an LOD of 2.0 pg/mL for the free PSA in 500 μL of serum per assay, using a self-created SPFS optical setup and peristaltic pump.16 Additionally, on the basis 1800

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Table 1. Kinetic Affinity Analysis of Desialyl-THGP of GalNAc Specific Lectins Including TJA-II, VVL, SBA, and WFA Using SPR Spectrometry at 25°Ca lectin material

ka (1/Ms)

TJA-II VVL SBA WFA

× × × ×

3.54 6.52 1.39 1.33

2

10 104 105 105

kd (1/s) 2.88 2.20 2.79 1.06

× × × ×

−3

10 10−3 10−3 10−3

KD (M) 8.14 3.38 2.01 8.00

× × × ×

10−6 10−8 10−8 10−9

a Affinity parameters ka, kd, and KD refer to the mean association rate constant, dissociation rate constant, and dissociation constant, respectively.

specificity or sensitivity of WFA itself or AF647-labeled WFA, on the basis of the SPR results (data not shown). Next, we used PSA secreted from LNCaP cells as a standard material for LacdiNAc-PSA. The LacdiNAc-PSA concentration in the culture medium from LNCaP cells was measured by WFA agarose column chromatography combined with total PSA ELISA, as previously reported.30 Interestingly, 55% of the total PSA in the culture medium from LNCaP cells was modified to LacdiNAc-PSA (data not shown).30 A standard curve was generated by an assay with varying concentrations of LacdiNAc-PSA in 1% BSA-PBS solution, as shown in Figure 5. The calibration curve showed that the LOD of LacdiNAc-PSA using the automated SPFS immunoassay was 20.0 pg/mL.

Figure 4. Calibration curves of total PSA in female serum by the SPFS immunoassay (●) and ELISA (△). The dotted-line represents the SPFS assay blank (mean = 1108 cps, SD = 61 cps). The ELISA assay blank was 3.022 AU (SD = 0.282 AU).

of the calibration curves, the SPFS immunoassay showed a wide dynamic range of at least five digits. Therefore, these results indicated that the volume of immobilized antibody on the plastic sensor chip was sufficient to quantify not only total PSA but also other commercial biomarkers for clinical diagnosis. Such a high-sensitivity total PSA immunoassay is indispensable for detection of relapse in patients with PC whose total PSA values have dramatically decreased following treatment with radiotherapy with or without hormonal therapy.34 This total PSA immunoassay system with both high sensitivity and high specificity should be further improved for the realization of personalized medicine for patients with PC and BPH. Calibration Curves of LacdiNAc-PSA by Sandwich Immunoassay Using WFA and Anti-PSA Monoclonal Antibody with SPFS. The total PSA immunoassay with SPFS clearly showed higher sensitivity at a lower reaction temperature of 25 °C, in comparison with CLEIA at 37 °C. This performance at low temperatures is the minimum condition necessary for a high-sensitivity lectin sandwich immunoassay because the ability of lectin to bind to the specific glycan motifs on the target glycoprotein increases under low temperature. We therefore investigated whether a lectin sandwich immunoassay with SPFS using a disposable sensor chip could be applied to quantitative measurement of LacdiNAc-PSA. The quantity of Fucα1−2Galβ and GalNAcβ1−4GlcNAc residues in total PSA could be detected in patients with PC but not in patients with BPH using TJA-II-Sepharose columns combined with total PSA ELISAs, as previously reported.30 However, TJA-II was difficult to apply using a two-step sandwich lectin immunoassay due to the high dissociation constants (Table 1). Thus, kinetic affinity analysis of β-GalNAc reactive lectins, including TJA-II, VVL, SBA, and WFA, was performed using SPR spectrometry at 25 °C. On the basis of the kinetic values (ka, kd, and KD) of these lectins with respect to desialyl THGP (see Table 1), we found that WFA was the most promising and useful β-GalNAcspecific lectin applicable to the two-step LacdiNAc-PSA immunoassay. We also determined that the most appropriate temperature for the WFA immunoassay was about 25 °C based on the temperature dependency of the interaction between WFA and desialyl THGP, as determined using SPR (data not shown). Additionally, we could not show any difference in the

Figure 5. Calibration curve of LacdiNAc-PSA using the two-step sandwich automated SPFS immunoassay with fluorescently labeled WFA. The dotted line represents the assay blank (mean = 140 cps, SD = 71 cps).

Structural changes in the carbohydrate moieties of various glycoproteins have been reported to be induced by various tumor types. The structural changes in the N-glycans of carcinoma embryonic antigen (CEA) in colon cancers,35,36 cholinesterase37 and transferrin38 in hepatocellular carcinomas, and human chorionic-gonadotropin (hCG)39−41 and alkaline phosphatase (ALP)41 in choriocarcinomas were previously reported using various forms of lectin-Sepharose column chromatography combined with ELISA or determination of enzymatic activities.42 These reported biomarkers are expected to play crucial roles in next-generation diagnostics, which focus on the sugar chain characteristics of the glycoprotein molecule. However, since previously reported lectin-Sepharose column assays for new glycoprotein biomarkers often require prolonged assay times and large amounts of lectin materials (i.e., a few milligrams of lectin per column), lectin-Sepharose column chromatography has encountered a bottleneck in terms of its applications in clinical diagnoses. In contrast, the SPFS lectin 1801

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Analytical Chemistry immunoassay uses about 100 ng of lectin per assay as the detection reagent. Therefore, the result of the LacdiNAc-PSA calibration curve in Figure 5 clearly showed that the highly sensitive lectin sandwich SPFS immunoassay could serve as an effective and useful system, facilitating the clinical application of other valuable diagnostic glycoprotein biomarkers. Furthermore, it is expected that the SPFS system will be applicable not only to clinical diagnosis but also to academic research. Serum Content of LacdiNAc-PSA in Patients with PC and BPH, as Determined by WFA Sandwich Immunoassay with SPFS Using a Plastic Sensor Chip. Finally, we evaluated the performance of the SPFS system for detection of LacdiNAc-PSA and compared this with the performance of ELISA for detection of total PSA using gray zone sera (total PSA of 4−20 ng/mL) from patients with PC (N = 44) and BPH (N = 27). The total PSA determination could not discriminate between PC and BPH because of its poor specificity (p = 0.2603), as shown in Figure 6a. Conversely,

Figure 7. Receiver operating characteristic (ROC) curve analysis for LacdiNAc-PSA (●) and total PSA (△).

the automated SPFS immunoassay for detection of LacdiNAcPSA will be further improved by reducing the influence of nonspecific reactions between fluorescently labeled WFA and serum proteins, we are investigating this process further. In a comparison with recently reported PSA assays, LacdiNAc-PSA was significantly better than pro-PSA,27 the 4k-panel,28 and free PSA.29 Additionally, it was similar in performance to the detection of Sia-2,3 PSA by fluorescent signals using a Luminex flowmetry system and anti-Sia-2,3 conformation IgG.43 In this study, we showed that the simple use of lectin for analysis of changes to carbohydrate structures was possible by employing highly sensitive SPFS lectin immunoassay technology. These results indicate that the SPFS system is a promising technique for the validation of new biomarkers (particularly those utilizing structural changes in carbohydrates) with applications in clinical diagnostics.

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Figure 6. Plot of the measured values of total PSA (a) using ELISA and LacdiNAc-PSA (b) with automated SPFS lectin immunoassays for PC (N = 44) and BPH (N = 27) in the gray zone (total PSA concentration: 4−20 ng/mL).

CONCLUSIONS The sandwich lectin immunoassay using SPFS with a disposable plastic sensor chip described in this study may be broadly applicable to clinical diagnosis of PC and other diseases using AF647-labeled lectins with higher sensitivity. On the basis of the results of this study, SPFS technology may be applicable for the validation of biomarkers and the detection of low levels of biomarkers in clinical samples.

the LacdiNAc-PSA determination (using SPFS) revealed that levels of LacdiNAc-PSA were significantly higher in PC sera than in BPH sera (p = 0.0025), as shown in Figure 6b. The results of receiver operating characteristic (ROC) curve analysis are shown in Figure 7. The area under the ROC curve for LacdiNAc-PSA (0.851) was significantly greater than that for total PSA (0.559) in this study. The optimum cutoff point giving low false positivity (40.7%) and high sensitivity (88.4%) was chosen, and the SPFS signal at this cutoff was 550 cps; this value was close to the average (+ SD) of normal sera. These results indicated that the performance of the automated SPFS immunoassay for LacdiNAc-PSA, using disposable plastic sensor chips, allowed for the discrimination between PC and BPH when the total PSA was in the gray zone. Although the number of clinical samples used in this study was small, the diagnostic value of LacdiNAc-PSA could be further and more appropriately evaluated by increasing the number of PC and BPH sera containing not only PSA in the gray zone but also total PSA exceeding 20 ng/mL. Evaluations under conditions in which PSA exists in excess are also important for understanding of influence of the inhibiting immunoreaction by nonLacdiNAc-PSA as well as the clinical significance of LacdiNAc-PSA. Since it is expected that the performance of

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by Konica Minolta Inc., Tokyo, Japan. REFERENCES

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DOI: 10.1021/ac503735e Anal. Chem. 2015, 87, 1797−1803