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Magnetically promoted rapid immunoreactions using functionalized fluorescent magnetic beads: a proof of principle.

Biomarkers provide information about disease progression and prognosis (1-4). They are also widely used in the diagnosis of disease, often at the point of care. Immunoassays are commonly used in the measurement of biomarkers in both the research and clinical arenas (5, 6). Although ELISA is commonly used (7, 8), it is a time-consuming and relatively complex approach. Thus, platforms that shorten assay time and reduce the number of steps involved would be beneficial. Immunoreaction using magnetic particles has attracted attention as a means to improve assay efficiency (9, 10). Reports of immunoassays with magnetic particles have mainly involved magnetically assisted separation of antibody-coated magnetic particles from reaction medium to facilitate assay processes. Here we describe a magnetically prompted immunoreaction system with unique polymer-coated magnetic beads containing highly analytically sensitive fluorophores. The immunoassay and the immunohistochemical staining described here feature simple magnetic collection of the beads in a specific space and require no signal amplification step for the detection of biomarkers, thus enabling the analytically sensitive and rapid detection of disease-related biomarkers.

Materials and Methods

Affinity magnetic beads with carboxylic acid were prepared according to previously reported procedures (11,12). Eu[(TTA).sub.3][(TOPO).sub.2] [thenoyltrifluoroacetylacetone (TTA) [3]; tri-n-octylphosphine oxide (TOPO)] was prepared from europium(III) acetate hydrate (Sigma), TTA, and TOPO (13). Transmission electron microscope (TEM) images were measured by JEM-2010F (JEOL) at the Center for Advanced Material Analysis, Tokyo Institute of Technology. Brain natriuretic peptide (BNP) and anti-BNP antibodies (KY-BNP-II and BC203) were kind gifts from Shionogi & Co. Prostate-specific antigen (PSA) and anti-PSA antibodies (5A6 and 1H12) were obtained from HyTest. A magnetic plate with 96 magnets was obtained from OZ Biosciences. For the comparison study, the Access Hybritech PSA (Beckman Coulter) was used for measurement of PSA concentrations. Anti-epidermal growth factor receptor (EGFR) antibodies were obtained from the hybridoma cell line 528 (HB-8509), which produces mouse monoclonal antibody against human EGFR, and purified using the Mab Trap[TM] kit (GE Healthcare). Anti-epithelial cell adhesion molecule (EpCAM) and anti-CA19-9 antibodies were obtained from Abcam. Epidermoid carcinoma cells A431 (CRL1555) and small-cell lung cancer cells H69 (HTB-119) were obtained from ATCC. The A431-GFP stable cell line was obtained by transfection of pEGFP-N1 (Promega) followed by G418 selection. A431 cells and H69 cells were maintained in DMEM and in RPMI 1640 supplemented with 10% fetal bovine serum in a 5% C[O.sub.2] humidified incubator at 37[degrees]C. A431 or H69 cells (1 x [10.sup.6] cells) in 100 [micro]L PBS were subcutaneously implanted into nude mice in 2 regions of the upper back. When the tumors reached approximately 5 mm in diameter, they were removed and one was fixed in 10% neutral buffered formalin for 24 h. Fixed tumors were processed in a tissue processor and embedded in paraffin. The second tumor was embedded in optimum cutting temperature (OCT) compound and stored at -80[degrees]C.

Experiments on immunohistochemical staining were approved by the institutional ethics committee and were conducted in accordance with the guidelines of the Declaration of Helsinki. Informed consent was obtained from each patient. Needle-core biopsy samples were obtained from breast cancer patients (2006-2009). Samples from esophageal cancer patients undergoing surgery (2001-2006) were used for EpCAM and cytokeratin 19 staining.

PREPARATION OF FLUORESCENT FERRITE BEADS

A suspension of affinity magnetic beads with carboxylic acid (1.0 mg) was incubated with 10 mmol/L Eu[(TTA).sub.3][(TOPO).sub.2] solution in acetone in the dark with vigorous shaking for 1 h at room temperature. Distilled water was added and acetone was evaporated under vacuum at 60[degrees]C. The pellet was washed with washing buffer [50 mmol/L HEPES (pH 7.9), 0.1% NP-40], and the prepared fluorescent ferrite (FF) beads were dispersed in distilled water and stored in the dark at 4[degrees]C.

PREPARATION OF ANTI-BNP ANTIBODY-COATED FF BEADS

A suspension of FF beads with carboxylic acid (1.0 mg) in distilled water was incubated with 20 g/L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC x HCl) in distilled water for 30 min at 4[degrees]C. The activated FF beads were washed with 2-morpholinoethanesulfonic acid-sodium hydroxide (MES-NaOH) (pH 6.0) and were incubated with 1.0 g/L anti-BNP antibody [F[(ab').sub.2] fragment of KY-BNP-II] in MES-NaOH (pH 6.0) for 2h at 4[degrees]C. Then, 1.0 mol/L ethanolamine solution in MES-NaOH (pH 6.0) was added, and the suspension was mixed overnight at 4[degrees]C. Finally, anti-BNP antibody-coated FF beads were washed and stored in HEPES buffer [10 mmol/L HEPES (pH 7.9), 50 mmol/L KCl, 1.0 mmol/L EDTA, 0.1% Tween 20] at 4[degrees]C.

PREPARATION OF ANTI-PSA ANTIBODY-COATED FF BEADS

A suspension of FF beads with carboxylic acid in PBS (pH 7.4) was incubated with 200 mmol/L EDC x HCl in PBS and 200 mmol/L N-hydroxysuccinimide in PBS for 4h at 4[degrees]C. The activated FF beads were washed with acetate (pH 5.2) and were incubated with 1.0 g/L anti-PSA antibody (5A6) in acetate for 1h at 4[degrees]C. Then, 1.0 mol/L ethanolamine solution in PBS was added and the suspension was mixed for 1 h at 4[degrees]C. Finally, anti-PSA antibody-coated FF beads were washed and stored in HEPES buffer at 4[degrees]C.

PREPARATION OF ANTI-EGFR, ANTI-EpCAM, AND ANTI-CA19-9 ANTIBODY-COATED FF BEADS

For the preparation of anti-EGFR, anti-EpCAM, and anti-CA19-9 antibody-coated FF beads, the procedure was the same as the procedure for anti-BNP antibody-coated FF beads. Antibody-coated FF beads were stored in HEPES buffer at 4[degrees]C.

MAGNETICALLY INDUCED SANDWICH IMMUNOASSAY WITH FF BEADS FOR DETECTION OF BNP

Polystyrene 96-well microplates (Matrix Technologies) were coated with 10 mg/L anti-BNP antibody (BC203) in carbonate (pH 9.6) overnight at 4[degrees]C. After removal of carbonate, blocking solution (10 mmol/L HEPES (pH 7.9), 50 mmol/L KCl, 1.0 mmol/L EDTA, 0.1% Tween 20, 1% skim milk) was added. BNP in reaction buffer (10 mmol/L HEPES (pH 7.9), 50 mmol/L KCl, 1.0 mmol/L EDTA, 0.1% Tween 20, 1% BSA) was added to each well and incubated for 20 s at 4[degrees]C. Next, 50 mg/L anti-BNP antibody (KY-BNP-II)-coated FF beads in reaction buffer (20 [micro]L) were added, the plate was gently shaken for 10 s, and placed on the magnet plate for incubation times of 0.5-3 min. The wells were then washed with reaction buffer and time-resolved fluorescence measurements were performed using a plate reader (ArvoSx1420; PerkinElmer).

MAGNETICALLY INDUCED SANDWICH IMMUNOASSAY WITH FF BEADS FOR DETECTION OF PSA

Polystyrene 96-well microplates were coated with 5.0 mg/L anti-PSA antibody (1H12) in carbonate (pH 9.6) overnight at 4[degrees]C. After removal of carbonate, blocking solution [25 mmol/L Tris-HCl (pH 7.9), 150 mmol/L KCl, 5.0 mmol/L EDTA, 0.1% Tween 20, 1% skim milk] was added. PSA in blocking solution was added to each well and incubated for 3 min at 4[degrees]C. Next, 12.5 mg/L anti-PSA antibody (5A6)-coated FF beads in blocking solution (20 [micro]L) were added, and the plate was placed on the magnet plate for incubation times of 1-30 min. The wells were then washed with blocking solution and time-resolved fluorescence measurements were performed using a plate reader (ArvoSx1420; PerkinElmer).

IMMUNOHISTOCHEMICAL STAINING OF PARAFFIN-EMBEDDED SAMPLES

Paraffin sections (4.0 [micro]m thick) were mounted on slides, deparaffinized in xylol, and rehydrated in a descending ethanol series for immunostaining. Rehydrated samples were treated with proteinase K (0.4 g/L; Dako) for 30 min, incubated with 0.5% periodic acid for 10 min at room temperature, washed with water, and then blocked with 4% Block Ace (Snow Brand Milk Products) in Tris-buffered saline (TBS) for 30 min at 37[degrees]C. After removal of the blocking solution, samples were incubated with primary antibody in blocking solution overnight at 4[degrees]C, washed with TBS, and then incubated with ENVISON (Dako) for 30 min. After washing, images were visualized using 3,3'-diaminobendizine (DAB), with hematoxylin counterstaining for 1 min. OCT-embedded frozen samples were sectioned on a cryostat microtome and dried for 1 min at 60[degrees]C, fixed with cold acetone for 2 min, hydrated, and then treated with blocking solution.

RAPID IMMUNOHISTOCHEMICAL STAINING OF TISSUES USING FF BEADS

Samples were treated with 5% BSA blocking solution for 2 min. After removal of blocking solution, samples were incubated with antibody-coated FF beads with a magnet beneath for 10 min, washed with TBS with gentle movement of the magnet, and examined using a fluorescence microscope (ECLIPS E1000; Nikon). OCT-embedded samples were sectioned on a cryostat microtome, dried for 1 min at 60[degrees]C, fixed with cold acetone for 2 min, hydrated, and treated with blocking solution.

Results

PRODUCTION OF UNIQUE 140-nm-DIAMETER FF BEADS

We previously developed unique multifunctional beads of 200 nm in diameter in which both fluorophores and ferrite nanoparticles were completely encapsulated into the organic polymer (14). In light of the beads' features, we expected that the FF beads would have the potential to be a practical probe in an immunoassay. Meanwhile, we also created 140-nm-diameter affinity magnetic beads (12). Because the 140-nm beads were more magnetically responsive than the 200-nm-diameter beads (11,15), we prepared 140-nm-diameter FF beads. We assumed that the use of hydrophilic fluorophores would result in fluorophore leakage from the beads and would complicate assay outcomes. Thus, the hydrophobic fluorescent complex Eu[(TTA).sub.3][(TOPO).sub.2] (16-19) was selected as the fluorophore for encapsulation into the polymer layer of the affinity magnetic beads. Using methanol to encapsulate Eu[(TTA).sub.3][(TOPO).sub.2] into the beads, we successfully produced 140-nm-diameter FF beads (Fig. 1A). TEM images showed that the shape of the FF beads was exactly the same as that of the affinity magnetic beads and the beads suffered no serious damage despite the use of organic solvents in the process of incorporating fluorophores into the beads (see Fig. 1A in the Data Supplement that accompanies the online version of this report at http://www.clinchem.org/content/ vol60/issue4). The FF beads dispersed well in PBS, in which they appeared as a dark-brownish suspension (derived from the ferrite nanoparticles) under visible light. Under ultraviolet exposure, the suspension emitted bright red fluorescence derived from naked Eu[(TTA).sub.3][(TOPO).sub.2]. Dispersed FF beads were collected easily using a permanent magnet and only the accumulated pellets showed strong fluorescence under ultraviolet irradiation (Fig. 1B). No leaking of fluorophores from FF beads into PBS was observed during many repeated collections of the magnetic beads.

MAGNETICALLY PROMPTED RAPID SANDWICH IMMUNOASSAY USING FF BEADS

Our sandwich immunoassay with FF beads is illustrated in Fig. 2. Detection antibody-coated FF beads and samples were added on a capture antibody-coated microplate, a magnet was attached under the plate for 1-2 min to concentrate the FF beads onto the immobilized capture antibody, unbound FF beads were washed out (similar to standard immunoassays), and the fluorescence of the remaining FF beads held on the plate through the antigen-antibody reaction was measured directly.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

To verify the feasibility of the newly devised system, we examined a sandwich immunoassay with FF beads using BNP, which is a hormone secreted by the heart and a basic biomarker of heart failure, as the target antigen (20, 21). The BNP concentration in the blood plasma of healthy persons is usually <20 pg/mL, and concentrations of >100 pg/mL BNP are associated with heart disease (22, 23). Magnetic collection of anti-BNP antibody-coated FF beads in an immunoreaction allowed dose-dependent detection of BNP within 1 min, after which saturation was observed (Fig. 3A). Although background fluorescence was observed in the absence of BNP, antigen concentrations equivalent to 2.0 pg/mL could be detected with a high signal-to-noise ratio. By contrast, the assay without magnetic collection of FF beads could detect only 200 pg/mL BNP (Fig. 3B). Without magnets, the fluorescence intensity derived from the FF beads held on the plate through the antigen-antibody reaction was highly variable. Fig. 3, C-E, show the detection of BNP at single incubation times of 1, 2, and 3 min. These graphs indicate that the magnetic force clearly enhanced the detection of BNP. Notably, a specific antigen-antibody reaction could be detected rapidly by only 1-min magnetic collection of FF beads without the use of any enzyme and enzymatic reaction.

We next investigated detection of PSA, a widely used biomarker in patients with prostate cancer (24). Healthy individuals generally have low concentrations of PSA (<0.1 ng/mL), whereas patients with prostate cancer have concentrations >4.0 ng/mL; hence, clinical examination of prostate cancer requires detection of PSA in serum ranging from 0.1 to 10 ng/mL (25, 26). PSA could be detected using anti-PSA antibody-coated FF beads either with or without magnetic collection of the beads (Fig. 4); however, there was a large difference in detection capability. With magnetic collection of FF beads, a wide range of PSA concentrations could be monitored (Fig. 4A). By contrast, in all cases in which magnetic collection was not used, the detection signal was less stable than the cases with magnetic collection (Fig. 4B). Using the magnetic system, we successfully detected as little as 0.02 ng/mL PSA within 5 min of sample addition. Fig. 4, C-F, show the detection of PSA at single incubation times of 1, 3, 10, and 30 min. As is the case in BNP, these graphs indicate that the magnetic force clearly enhanced the detection of PSA. On receiving the results, the performance of the sandwich immunoassay with FF beads was compared with conventional immunoassays. Using PSA samples whose concentrations had been measured by chemiluminescent EIA (CLEIA), we measured over 50 PSA samples by the sandwich immunoassay with FF beads (Fig. 5). Good correlation was observed between PSA concentrations measured by the sandwich immunoassay with FF beads and PSA concentrations measured by CLEIA. The correlation between the 2 assays was: y = 0.472 + 0.879x, [r.sup.2] = 0.953.

[FIGURE 3 OMITTED]

LIMIT OF DETECTION, LIMIT OF QUANTIFICATION, AND REPRODUCIBILITY IN THE SANDWICH IMMUNOASSAY WITH FF BEADS

The limit of detection (LOD) was defined as the lowest measured value for which the measured value minus 2 SDs was higher than the blank mean value plus 2 SDs. Based on this criterion, the LOD of the sandwich immunoassay with FF beads was estimated to be around 5.0 pg/mL for BNP in plasma and around 0.005 ng/mL for PSA in serum (see online Supplemental Figs. 2 and 3). The limit of quantification (LOQ) was defined as the lowest concentration measurable at an interassay CV <20% in the sandwich immunoassay with FF beads. Based on this criterion, the LOQ of the sandwich immunoassay with FF beads was estimated to be 10 pg/mL for BNP in plasma and 0.02 ng/mL for PSA in serum (see online Supplemental Figs. 2 and 3). For reproducibility of the sandwich immunoassay with FF beads, the mean value, SD, and CV were calculated. For the medium PSA concentration, the mean value, SD, and CV were 5.63 pg/mL, 0.489 pg/mL, and 8.7%, respectively. For the high PSA concentration, the mean value, SD, and CV were 10.1 pg/mL, 1.35 pg/mL, and 13.3%, respectively.

[FIGURE 4 OMITTED]

MAGNETICALLY PROMPTED RAPID IMMUNOHISTOCHEMISTRY USING FF BEADS

The scheme of an advanced immunohistochemical staining using magnetic collection of FF beads is illustrated in Fig. 6. FF beads coated with antibodies recognizing specific carcinoma cell-surface antigens are added onto fixed samples of carcinoma cells, and a magnet is attached beneath them to enhance the antigen-antibody reaction (10 min). After removal of the magnet, the samples are washed and observed directly by fluorescence microscopy. In this system, washing, a key step in immunohistochemical staining to eliminate undesired signal noise, was simplified by the use of a magnet.

[FIGURE 5 OMITTED]

To confirm the magnetically promoted immunohistochemical staining with FF beads, we selected xenograft samples of A431 human epidermoid cancer cells, in which EGFR is highly expressed. EGFR is a key disease marker to understand various types of malignancy, including breast cancer, because it plays an important role in tumor cell survival and proliferation (27, 28). Fig. 7A shows the results of immunohistochemical staining of A431 cells by hematoxylin--eosin (HE) staining, conventional polymer-labeled immunostaining with DAB, and anti-EGFR antibody-coated FF beads. Samples treated with FF beads showed vivid red florescence, and the areas of staining corresponded to those identified by the conventional polymer-labeled method. By contrast, samples prepared from H69 cells (small-cell lung cancer cells, EGFR expression level is relatively low) treated with anti-EGFR antibody-coated FF beads produced negligible levels of red fluorescence (Fig. 7B).

[FIGURE 6 OMITTED]

[FIGURE 7 OMITTED]

Next, immunohistochemical staining of breast cancer needle-core biopsies was investigated. Similar to the results with A431 cells, clear red fluorescence derived from FF beads was observed in the same area stained using the polymer-labeled method (Fig. 7C). These results indicate that antibody-coated FF beads can accurately and rapidly recognize corresponding antigens expressed on cells and tissues through a magnetically enhanced antigen-antibody reaction.

Discussion

Conventional immunoassays often exhibit low-level signals due to the use of enzyme-modified detection antibodies. Investigators have been trying to resolve the intrinsic problems of analytical sensitivity; for example, immunoassay systems using antibody-coated nanoparticle doping enzymes instead of enzyme-bound antibodies provide improvements in analytical sensitivity (29). These types of immunoassays use enzymes in the detection of biomarkers and take several hours to complete testing.

Magnetic particles are attractive functional materials in the development of immunoreactions, because magnetic force often improves efficiency and analytical sensitivity (30). Various types of immunoassays with magnetic particles have been developed and some have enabled shorter assay times (31-33). Mirkin and coworkers reported an analytically sensitive immuno-PCR method using magnetic microbeads coated with monoclonal antibodies to PSA as well as gold nanoparticles functionalized with bar-coded double-stranded DNA and polyclonal antibodies (34). However, the immuno-PCR method with magnetic microbeads and gold nanoparticles still requires several steps from sample addition to signal acquisition and takes at least 1.5-2 h for detection of PSA. Although there are highly analytically sensitive immunoassays that simply involve magnetic collection of functionalized magnetic particles, construction of a rapid immunoassay (<10 min) or assays without amplification steps has not previously been achieved. Similarly, conventional immunohistochemical staining generally requires overnight incubation of samples with primary antibodies for high analytical sensitivity. Staining of sentinel lymph node samples using pigments, including polymer-embedded staining, has been used for intraoperative diagnosis; however, pathological diagnosis with pigments is time-consuming and occasionally leads to misdiagnosis as a result of human error.

The FF beads that we have developed are unique materials in that both hydrophobic fluorophores and ferrite nanoparticles are completely encapsulated into an organic polymer that can be modified chemically (11, 15). The lack of fluorophore leakage from FF beads during magnetic separation indicates that all fluorophores are retained firmly inside the polymer of the beads. The FF beads were quite stable and could be stored for at least 6 months without any damage and loss of fluorescence intensity. In addition, the fluorescence intensity of FF beads changed depending on the concentration of the beads (see online Supplemental Fig. 1B). Thus, the FF beads should be highly advantageous for their application as probes in analytically sensitive bioassays capable of detecting specific biomarkers. The results of the sandwich immunoassay, using FF beads on which antibodies were coated, clearly demonstrate that magnetic collection of FF beads in the immunoreaction step enabled the detection of disease markers within 5 min after sample addition (Figs. 3A and 4A). In addition, the data of the biomarker detection at a single incubation time indicated that several minutes of magnetic collection of FF beads markedly improved the detection capability (Fig. 3, C-E, and Fig. 4, C-F). Although a 3-10-min immunoreaction with the magnet seemed to be most effective for the detection of the biomarkers by the sandwich immunoassay with FF beads, we recognized that 1-min immunoreaction with the magnet was enough for the detection of the biomarkers. Additionally, the results of the immunohistochemical staining with antibody-coated FF beads also demonstrated that a 10-min magnetic collection of FF beads in the incubation step enabled the ultrarapid and reliable discrimination of carcinoma cells (Fig. 7). By contrast, without magnetic collection, both immunoassays resulted in low detection of disease markers and required the same time as conventional methods to obtain comparable analytical sensitivity (Figs. 3B and 4B). Furthermore, we also found that under the same conditions, including the assay time, disease-related markers in the plasma or serum could be detected without decreases of florescence counts (see online Supplemental Figs. 2 and 3). These results clearly indicate that the magnetic force enhances and accelerates specific interactions between antigens and antibodies, and the application of magnetic collection of FF beads in a particular space to the immunoreaction can dramatically improve both the total assay time and the number of steps required, regardless of the antibodies used and the presence of the plasma or the serum.

The LOQ values of 10 pg/mL for BNP and 0.02 ng/mL are comparable to those for currently used immunoassays. The relatively low LOQ indicates that the sandwich immunoassay with magnetic collection of FF beads is a practical and reliable immunoassay system with sufficient analytical sensitivity. Often the analytical sensitivity of immunoassays depends on antibody quality (35). We speculate that the difference of detection pattern between BNP and PSA would result from differences in the quality of the antibodies used in the sandwich immunoassay with FF beads.

The successful rapid and analytically sensitive detection of target molecules (BNP, PSA, and EGFR) by magnetic collection of FF beads in the immunoreaction steps can be explained by the magnetic concentration of both antigens and antibodies locally on the plate or samples, followed by direct florescent measurement without the need for signal amplification. The results suggest that the magnetic concentration of biomoleculecoated FF beads to a specific place has the potential to accelerate the interaction between biomolecules. We also investigated the versatility of the immunohistochemical staining with FF beads using several carcinoma cells and found that carcinoma cells other than A431 cells could be discriminated within 20 min (see online Supplemental Fig. 4).

To date, several immunoreaction platforms that use magnetic force to improve their efficiency have been reported (30-33). These mainly involve removal of unbound capture antibodies and, to the best of our knowledge, there are few bioassays that accelerate specific biomolecule interactions and shorten assay times using magnetic force (36). The immunoreaction systems with FF beads described here used magnetic force, not to remove the magnetic beads but to enhance the signal for detection of biomarkers. The simple operation of collecting FF beads magnetically at a specific space in the immunoreaction enables improvements in both analytical sensitivity and total assay time. Compared to conventional platforms, the advanced immunoreaction with FF beads enables rapid detection of biomarkers without the use of enzyme reactions.

In summary, we successfully developed a rapid immunoreaction system using uniquely developed fluorescent magnetic beads, FF beads. Two distinguishing properties of FF beads, strong magnetic force and high fluorescence intensity, enable the shortening of assay time dramatically without loss of sensitivity and eliminate the signal amplification step. Immunoreaction systems with FF beads would be feasible alternatives to conventional immunoassays and immunohistochemical staining.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, oranalysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: Y. Yamaguchi, Grant-in-Aid for Scientific Research on Innovative Areas "Transcription Cycle" from MEXT; Y. Kiatagawa, Grant for Research and Development Projects in Cooperation with Academic Institutions from the New Energy and Industrial Technology Development Organization (NEDO); H. Handa, Grant for Research and Development Projects in Cooperation with Academic Institutions from the New Energy and Industrial Technology Development Organization (NEDO), Grant-in-Aid for Scientific Research on Innovative Areas "Chemical Biology of Natural Products" from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Grant-in-Aid for Scientific Research (A) from Japan Society for the Promotion of Science (JSPS), Health Labour Sciences Research Grants (Research on New Drug Development) from the Ministry of Health, Labour and Welfare (MHLW), and Development of Systems and Technologies for Advanced Measurement and Analysis from the Japan Science and Technology Agency (JST).

Expert Testimony: None declared.

Patents: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We deeply thank Shionogi & Co. for the kind gift of BNP-related materials. We also thank Prof. Haruma Kawaguchi (Kanagawa University) for TEM observations and Naohiro Hanyu and Dr. Toshiyuki Tanaka (TamagawaSeiki) for fruitful discussions on immunohistochemical stainings.

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Satoshi Sakamoto, [1] ([dagger]) Kenshi Omagari, [2] ([dagger]) Yoshinori Kita, [1] Yusuke Mochizuki, [1] Yasuyuki Naito, [1] Shintaro Kawata, [1] Sachiko Matsuda, [2] Osamu Itano, [2] Hiromitsu Jinno, [2] Hiroya Takeuchi, [2] Yuki Yamaguchi, [1] Yuko Kitagawa, [2] and Hiroshi Handa [1] *

[1] Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan; [2] Department of Surgery, School of Medicine, Keio University, Tokyo, Japan.

[3] Nonstandard abbreviations: TTA, thenoyltrifluoroacetylacetone; TOPO, tri-n-octylphosphine oxide; TEM, transmission electron microscopy; BNP, brain natriuretic peptide; PSA, prostate specific antigen; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; OCT, optimum cutting temperature; FF, fluorescent ferrite; EDC-HCl, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; MES-NaOH, 2-morpholinoethanesulfonic acid-sodium hydroxide; TBS, Tris-buffered saline; DAB, 3,3'-diaminobendizine; CLEIA, chemiluminescent EIA; LOD, limit of detection; LOQ, limit of quantitation; HE, hematoxylin eosin.

([dagger]) Satoshi Sakamoto and Kenshi Omagari contributed equally to the work, and both should be considered as first authors.

* Address correspondence to this author at: Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. Fax +81-45-924-5834; e-mail hhanda@bio.titech.ac.jp.

Received July 10, 2013; accepted December 2, 2013.

Previously published online at DOI: 10.1373/clinchem.2013.211433
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Title Annotation:Automation and Analytical Techniques
Author:Sakamoto, Satoshi; Omagari, Kenshi; Kita, Yoshinori; Mochizuki, Yusuke; Naito, Yasuyuki; Kawata, Shi
Publication:Clinical Chemistry
Article Type:Report
Date:Apr 1, 2014
Words:5438
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