ORIGINAL ARTICLE

Generation of an anti-Dabigatran Monoclonal Antibody and Its Use in a Highly Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Serum Dabigatran Shigeru Oiso, PhD,*† Osamu Morinaga, PhD,*‡ Takaaki Goroku,† Takuhiro Uto, PhD,*‡ Yukihiro Shoyama, PhD,*‡ and Hiroko Kariyazono, PhD*†

Background: Dabigatran (DT) is a direct thrombin inhibitor used to prevent venous and arterial thromboembolism due to atrial fibrillation. DT is the active form of the commercially available prodrug DT etexilate. Although DT has many clinical advantages over warfarin, it increases the incidence of bleeding in patients with renal dysfunction. Circulating levels of DT are increased in such patients because it is mainly eliminated by renal excretion. Therapeutic drug monitoring may therefore help to prevent adverse DT effects, but no method for measuring circulating DT levels has been reported, except for an analysis by liquid chromatography– tandem mass spectrometry. This study sought to develop a novel enzyme-linked immunosorbent assay (ELISA) to measure DT concentrations.

Methods: Mice were immunized with a DT-keyhole limpet hemocyanin conjugate to generate an anti-DT antibody. Immunized mouse splenocytes and myeloma cells (SP2/0) were fused to obtain an anti-DT monoclonal antibody (DT-mAb). DT-mAb and DT solutions were added to microplate wells coated with a DT-human serum albumin conjugate. DT concentrations were determined based on the principles of ELISA. Results: DT-mAb was successfully purified from a hybridoma, and the competitive ELISA developed using this DT-mAb could evaluate DT concentrations ranging from 7.8 to 125 ng/mL. The ELISA signal was not linear using DT-spiked serum; however, it was linear when serum ultrafiltrate was used. Weak cross-reactivity with DT etexilate was detected, but no cross-reactivity was observed with other structurally related drugs or drugs commonly used for the treatment of atrial fibrillation. Conclusions: The developed competitive ELISA is a valuable and specific tool to analyze free DT in serum ultrafiltrate for therapeutic drug monitoring and pharmacokinetic studies.

Received for publication September 26, 2014; accepted December 23, 2014. From the *Graduate School of Pharmaceutical Sciences; Departments of †Pharmaceutical Health Care Sciences; and ‡Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagasaki International University, Japan. Supported by JSPS KAKENHI Grant Number 24659355. The authors declare no conflict of interest. Correspondence: Shigeru Oiso, PhD, Graduate School of Pharmaceutical Sciences, Department of Pharmaceutical Health Care Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch Sasebo, Nagasaki 859-3298, Japan (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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Key Words: dabigatran, monoclonal antibody, enzyme-linked immunosorbent assay, serum ultrafiltrate (Ther Drug Monit 2015;37:594–599)

INTRODUCTION Dabigatran (DT), the active form of DT etexilate, produces anticoagulant effects by direct inhibition of thrombin.1–3 DT prevents venous and arterial thromboembolism due to atrial fibrillation and was developed recently as a clinical alternative to warfarin, a vitamin K antagonist that has been widely used as an anticoagulant for decades.1,4–7 During warfarin treatment, it is necessary to carefully monitor the balance between thrombotic inhibition and the risk for bleeding. This is because warfarin can interact with diverse foods and drugs and has a slow onset and offset of action and a narrow therapeutic window.1,4,7 In contrast, DT has a rapid onset of action, its blood concentrations and anticoagulant effects are dose dependent and predictable, and it interacts with fewer drugs and food.1 For these reasons, routine coagulation monitoring is not considered necessary during DT treatment.1 Furthermore, DT is as effective as warfarin in the prevention of acute venous thromboembolism and stroke in patients with atrial fibrillation, with a significantly lower rate of bleeding.8,9 Although DT has many advantages over warfarin, a high incidence of bleeding in patients with renal dysfunction taking DT has been reported.1,10 This is due to decreased renal excretion, leading to enhanced blood DT concentrations.1,4 Therefore, the dosage of DT etexilate should be decreased in patients with renal dysfunction.1,11 Therapeutic drug monitoring (TDM) is useful to titrate appropriate dosages of drugs affected kinetically by renal dysfunction. As a method for measuring blood DT concentrations, the technique has already been reported by liquid chromatography–tandem mass spectrometry (LC–MS/MS).2 However, using LC–MS/MS in many medical institutions is difficult because of its expensiveness. Therefore, the development of a convenient and low-cost method for blood DT quantification would be advantageous. After administration of DT etexilate, the peak and trough levels of DT in the circulation at steady state were 184 and 37 ng/mL, respectively,11 indicating that a highly sensitive assay is required for effective TDM of DT. Enzymelinked immunosorbent assay (ELISA) is a sensitive method that has been widely used for the measurement of drug concentrations in the blood, and this method is much more Ther Drug Monit  Volume 37, Number 5, October 2015

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convenient and lower cost than LC–MS/MS. As ELISA uses antigen–antibody reactions, a specific antibody for the target compound is required. Low-molecular-weight compounds are not usually antigenic; therefore, it may be difficult to generate a specific antibody by immunizing animals with the compound alone. However, a technique has been developed for generating antibodies to low-molecular-weight compounds by immunization with a conjugate of the target compound and a carrier protein, such as keyhole limpet hemocyanin (KLH), human serum albumin (HSA), and bovine serum albumin (BSA). Antibodies produced in this manner have been applied to ELISA systems for many compounds, including medicines and plant components.12–15 In this article, we report the successful generation of a specific monoclonal antibody for DT (DT-mAb) after mouse immunization with a DT-KLH conjugate. The DTmAb was used to develop an ELISA for DT quantification. This ELISA may provide a useful approach for the measurement of free DT concentrations in serum sample ultrafiltrates.

MATERIALS AND METHODS Materials DT and DT etexilate were purchased from Toronto Research Chemicals (Toronto, Canada). Peroxidase-labeled anti-mouse IgG and N-hydroxysuccinimide (NHS) were purchased from Sigma (St. Louis, MO). 1-Ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC) was obtained from Dojindo (Kumamoto, Japan). KLH, HSA, 2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), allopurinol, disopyramide, indomethacin, propranolol hydrochloride, theophylline, and verapamil hydrochloride were obtained from Wako Pure Chemical Industries Ltd (Osaka, Japan). Amiodarone hydrochloride, candesartan cilexetil, lansoprazole, omeprazole, and telmisartan were purchased from Tokyo Chemical Industry (Tokyo, Japan). Digoxin was purchased from Nacalai Tesque (Kyoto, Japan). E-RDF (enriched RPMI 1640-Dulbecco–Ham F12) medium was obtained from Kyokuto Pharmaceutical Industrial Co Ltd (Tokyo, Japan). Protein G sepharose was purchased from GE Healthcare (Buckinghamshire, England). Human serum (Lot: H40412D) was purchased from Kohjin Bio (Tokyo, Japan).

Dabigatran Enzyme-Linked Immunosorbent Assay

DT-HSA was mixed with a 1000-fold molar excess sinapinic acid in an aqueous solution containing 0.15% (vol/vol) trifluoroacetic acid. The mixture was subjected to a JEOL mass spectrometer time-of-flight mass monitor and irradiated with an N2 laser (337 nm, 150 ns). The ion formed by each pulse was accelerated by a 20 kV potential into a 2.0-m evacuated tube.

Production of the Anti-DT Monoclonal Antibody DT-KLH conjugates (50 mcg) were dissolved in phosphate-buffered saline (PBS), emulsified in an equal volume of Freund complete adjuvant, and injected intraperitoneally into BALB/c male mice (6 weeks old). Immunization was repeated 2 weeks later using the same amount of immunogen emulsified in Freund incomplete adjuvant. After an additional 2 weeks, the animals received an intraperitoneal injection of 25 mcg DT-KLH conjugates dissolved in PBS without adjuvant, and this was repeated a further 6 times at 2-week intervals. The anti-DT response in the immunized mouse serum was determined by direct ELISA, as described below. On the third day, after the final immunization, mouse splenocytes were fused with SP2/0-Ag14 myeloma cells using the polyethylene glycol method,17 and hybridomas were selected in e-RDF medium containing hypoxanthine (100 mmol/L), aminopterin (0.4 mmol/L), and thymidine (16 mmol/L). Hybridomas secreting DT-mAbs were cloned using the limiting dilution method,18 and DT-mAbs were purified using protein G sepharose. Culture media conditioned by hybridomas secreting DT-mAbs were loaded onto a column packed with protein G sepharose. The adsorbed DT-mAbs were eluted with 100 mmol/L citrate buffer (pH 2.7), followed by column washing with 20 mmol/L phosphate buffer (pH 7.0). The eluted DT-mAbs were neutralized with 1 mol/L Tris solution, dialyzed with water, and lyophilized.

Direct and Competitive ELISA

DT was conjugated with KLH and HSA according to a method described by Li et al.16 Briefly, DT (5 mg) and NHS (2.5 mg) were dissolved in dimethyl sulfoxide, and the solution was stirred for 15 minutes at room temperature. EDC (5 mg) in 100 mL of 0.1 mol/L 2-morpholinoethanesulfonic acid (MES) buffer (pH 4.7) was then added to the solution. After stirring for 15 minutes at room temperature, KLH (5 mg) or HSA (5 mg) in 1 mL of 0.1 mol/L MES buffer was added, and the mixture was stirred for 15 hours at 48C. The reaction mixture was dialyzed for 3 days and then lyophilized to produce DTKLH and DT-HSA conjugates. The hapten number in the DTHSA conjugate was calculated from the molecular weight, determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

The reactivity of DT-mAbs to DT, KLH, HSA, and BSA was determined by direct ELISA. DT-HSA, KLH, HSA, or BSA solution (100 mL of 1 mcg/mL in 50 mmol/L carbonate buffer, pH 9.6) were added to a 96-well microplate (Thermo Fisher Scientific, Waltham, MA) and incubated at 378C for 1 hour to coat each antigen onto the well surface. The wells were then blocked with 300 mL of 5% skimmed milk in PBS at 378C for 1 hour. After washing the plate 3 times with PBS containing 0.05% Tween-20 (T-PBS), 50 mL of mouse anti-DT serum solution (1/100 in T-PBS) or DTmAb solution (in T-PBS), and the same volume of purified water, was added to the wells and incubated at 378C for 1 hour. After washing 3 times with T-PBS, 100 mL of peroxidase-labeled anti-mouse IgG (1:1000) was added to each well and incubated for 1 hour at 378C. Each well was washed 3 times with T-PBS before adding 100 mL of substrate solution [200 mmol/L citrate buffer (pH 4.0) containing 0.003% H2O2 and 0.3 mg/mL ABTS] to each well and incubating for 20 minutes. The absorbance of each well was then measured at 405 and 490 nm using a microplate reader, and the difference in absorbance at these 2 wavelengths was used for subsequent analysis.

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Preparation of DT-protein Conjugates

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Oiso et al

For the competitive ELISA, 50 mL of various concentrations of DT dissolved in purified water, serum, serum ultrafiltrate, or Ringer solution was added to 50 mL of DT-mAb solution at the step where DT-mAb was added. The subsequent steps were then performed as described above. Serum ultrafiltrate was prepared by centrifugation of serum at 14,000g for 30 minutes using a Nanosep centrifugal device (Pall Corporation, WA).

DT Quantitation Using High-Performance Liquid Chromatography An LC-2000 HPLC system (JASCO, Tokyo, Japan) equipped with an ultraviolet detector was used for the measurement of DT concentrations. Samples were mixed with an equal volume of 1 mmol/L p-toluenesulfonic acid solution as an internal standard and separated on a 4.6 · 250 mm Cosmosil 5C18-MS-II packed column (Nacalai Tesque). The measuring conditions were examined based on analysis conditions by LC–MS/MS.2 The mobile phase consisted of aqueous ammonium formate (0.02 mol/L, pH 8.0) and ammonium formate in methanol (0.02 mol/L, pH 8.0) mixed at a ratio of 11:9 (vol/vol), with a flow rate of 0.5 mL/min and a detector wavelength of 254 nm. The data were analyzed using ChromNAV software (JASCO).

Cross-reactivity of the Anti-DT Monoclonal Antibody The cross-reactivity (CR) of the test compounds with DT-mAb was determined according to the equation by Weiler and Zenk.19 Briefly, the 50% inhibitory concentration (IC50) of DT and the test compounds for the binding of DT-mAb to DT were measured by competitive ELISA, as described above. The CR was calculated by the following equation: CRð%Þ ¼ ðIC50   of   DT=IC50   of   test  compoundÞ · 100:

RESULTS AND DISCUSSION Direct Determination of DT-HSA Conjugate by MALDI-TOF-MS DT does not have any immunogenicity because of its low molecular weight. To produce a specific antibody to DT, we first prepared immunogenic conjugates of DT and carrier proteins. The DT carbonyl group was linked to the amino groups of the carrier proteins KLH and HSA by an NHS ester-mediated reaction (Fig. 1). MALDI-TOF-MS can directly identify whether the hapten number in an antigen conjugate is suitable for immunization.20–22 This method was used to determine the

hapten number in the DT-HSA conjugate. A broad peak corresponding to the DT-HSA conjugate was observed around m/z 68,082 (data not shown). This indicated that at least 3 DT molecules were bound to the HSA molecule, as the molecular weights of DT and HSA are 471 and 66,458, respectively. This hapten number was considered sufficient for immunization, based on previous reports.13,23 Although it was impossible to measure the hapten number of DT-KLH because of its very large molecular weight, a greater number of DT molecules may bind to KLH rather than to HSA because KLH has more amino groups available for conjugate formation. On the basis of these findings, we immunized BALB/c mice with the DT-HSA and DT-KLH conjugates.

Production and Characterization of the Anti-DT Antibody The immune response to DT in serum from mice immunized with DT-HSA and DT-KLH was monitored by direct ELISA. In both the DT-HSA and DT-KLH immunization groups, the anti-DT titers of the antisera increased after iterative immunization. However, the DT-HSA immunization group also showed a strong immune response to HSA. After the eighth immunization, splenocytes from mice immunized with DT-KLH, but not from those immunized with DT-HSA, were fused with P2/0-Ag14 myeloma cells16,24 to generate hybridomas, producing a monoclonal antibodies reactive to DT. The DT-mAb was classified as IgG1 with a k chain. The reactivity of the DT-mAb to DT, KLH, HSA, and BSA was investigated by direct ELISA using a variety of DTmAb concentrations. DT-mAb bound to DT in a concentration-dependent manner but did not bind KLH, HSA, or BSA. The absorbance was 1.5 at a DT-mAb concentration of 0.2 mcg/mL (Fig. 2), and this concentration was selected for competitive ELISA testing.

ELISA Sensitivity and Recovery of DT in Serum Samples A competitive ELISA was performed using 0.2 mcg/mL DT-mAb and a standard solution of DT in purified water as the competitive reagent. A calibration curve was generated using DT concentrations of 3.91–500 ng/mL, as shown in Figure 3. Under these conditions, the full measuring range of the assay was between 7.81 and 125 ng/mL. We investigated whether the competitive ELISA could be used to determine serum DT concentrations. As shown in Figure 4, the competitive ELISA produced different signals for samples containing the same concentrations of DT prepared in purified water and in serum. As it has been reported that approximately 35% of DT in the blood is protein-bound,4 the lower amount of free DT because of protein binding may cause the different signals in serum and water samples.

FIGURE 1. Conjugation of DT with the carrier proteins KLH or HSA.

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FIGURE 2. Affinity of the DT-mAb for DT, KLH, HSA, and BSA. DT-HSA, KLH, HSA, or BSA was coated onto the microwell plates, and direct ELISA were performed using the indicated concentrations of DT-mAb.

Therefore, the competitive ELISA was also performed using ultrafiltrates of the serum samples (Fig. 4). These samples showed similar ELISA signals to the serum samples. DT was not absorbed by the ultrafiltration device (data not shown), suggesting that the DT-mAb used in the ELISA detected only free DT in the serum. To test this, we spiked DT into the serum ultrafiltrate and analyzed these samples using the competitive ELISA. This produced linear plots in parallel with the results obtained from standard solutions prepared

Dabigatran Enzyme-Linked Immunosorbent Assay

FIGURE 4. Effect of serum and ultrafiltration of serum on the DT competitive ELISA. Purified water, serum, or serum ultrafiltrate (as indicated) was spiked with the same concentration range of DT before analysis. The data are expressed as the mean 6 SD of 3 independent experiments. A is the absorbance in the presence of DT, and A0 is the absorbance in the absence of DT.

with purified water, although the line was shifted to the left (Fig. 4). As serum ultrafiltrate contains some electrolytes, we dissolved DT in Ringer solution, which has a similar electrolyte composition to serum, and performed the competitive ELISA. These measurements were more consistent with those of serum ultrafiltrate spiked with DT (Fig. 5), suggesting that

FIGURE 3. The DT calibration curve for the competitive ELISA. The indicated concentrations of DT were incubated with DTmAb (0.2 mcg/mL) in a 96-well plate precoated with DT-HSA. The data are expressed as the mean 6 SD of 3 independent experiments. A is the absorbance in the presence of DT, and A0 is the absorbance in the absence of DT.

FIGURE 5. Effect of Ringer solution on the competitive DT ELISA. Ringer solution or serum ultrafiltrate (as indicated) was spiked with the same concentration range of DT before analysis. The data are expressed as the mean 6 SD of 6 independent experiments. A is the absorbance in the presence of DT, and A0 is the absorbance in the absence of DT.

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Oiso et al

TABLE 1. Competitive ELISA Quantitation of DT in Spiked Serum Ultrafiltrate Samples Spiked Concentration (ng/mL)

Measurement (ng/mL)*

7.81 15.62 31.25 62.50 125.00 Average

7.18 14.74 32.79 68.67 126.19

6 6 6 6 6

0.85 0.67 6.52 2.32 3.49

Recovery (%) 91.4 94.4 104.9 109.8 100.9 100.4

TABLE 3. Variability of the DT Competitive ELISA CV (%) Intra-Assay (n = 5) Concentration of DT (ng/mL) 7.81 15.62 31.25 62.50 125.00

*Values are mean of 6 determinations 6SD.

ELISA Variability Reproducibility is an essential criterion used to judge the reliability of immunoassay data. Table 3 shows the assay variation profile obtained by competitive ELISA analysis of DT solutions prepared in Ringer solution and serum ultrafiltrate. Well-to-well (intra-assay) and plate-to-plate (interassay) coefficients of variation were satisfactory over a DT range of 7.81–125 ng/mL (Table 3). Generally, reproducibility is influenced by various factors such as the quality and quantity of the coated hapten and the consistency of the reagents and experimental conditions used. Each assay plate

TABLE 2. Comparison of Competitive ELISA and HPLC Determinations of DT Spiked in Serum Ultrafiltrate Measurement (mcg/mL)* ELISA 8.25 15.16 31.75 71.81 124.08

6 6 6 6 6

0.55 0.45 2.23 2.16 4.56

*Values are the mean of 3 determinations 6SD. HPLC, high-performance liquid chromatography.

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Ringer Solution

Serum Ultrafiltrate

2.37 2.63 1.42 2.72 4.17

2.00 3.35 2.05 2.87 2.52

4.06 3.49 5.43 4.97 6.67

3.59 6.01 6.45 5.10 4.42

should contain a standard curve to account for interassay variability.

ELISA Specificity The competitive ELISA specificity was evaluated by calculating the DT-mAb CR with various related compounds. The following test drugs were selected: DT etexilate (the DT prodrug); amiodarone, disopiramide, propranolol, verapamil, and digoxin (drugs used to treat atrial fibrillation); and omeprazole, lansoprazole, candesartan cilexetil, telmisartan, allopurinol, theophylline, and indomethacin (drugs containing similar chemical structures to DT). DT-mAb had 1.57% and 1.68% CR with DT etexilate in Ringer solution and serum ultrafiltrate, respectively, and no detectable CR with the other compounds tested (Table 4). This weak reaction with DT etexilate was due to its structural similarity to DT. However, as almost all of the administered DT etexilate molecules are converted to DT by esterase after oral administration,2,3 this slight CR would not affect the accuracy of this competitive DT ELISA in patients.

Limitations We have not yet confirmed the clinical utility of the developed ELISA using serum samples from DT-treated TABLE 4. DT-mAb Cross-reactivities CR (%) Compound

7.81 15.62 31.25 62.50 125.00

Serum Ultrafiltrate

CV, coefficients of variation.

electrolytes may influence the competitive ELISA. In this assay, the recovery values of the spiked DT ranged from 91.4% to 109.8%, with an average of 100.4% (Table 1). To confirm the accuracy of the competitive ELISA, highperformance liquid chromatography was also performed using serum ultrafiltrate spiked with 7.81–125 mcg/mL DT, and the measurements were compared with those of the competitive ELISA. Because the competitive ELISA had a higher sensitivity, the samples were diluted to within the assay range using Ringer solution. The concentrations detected by both methods were highly consistent (Table 2). These results suggested that the concentration of DT in serum ultrafiltrates could be measured accurately over the range of 7.81–125 ng/mL by the competitive ELISA using standard solution diluted in Ringer solution and indicated that this ELISA may be applicable for monitoring free DT concentrations in blood.

Spiked Concentration (mcg/mL)

Interassay (n = 4)

Ringer Solution

HPLC 8.39 15.72 30.65 61.54 126.52

6 6 6 6 6

0.32 0.13 0.11 0.31 0.58

Dabigatran Dabigatran etexilate Amiodarone Disopiramide Propranolol Verapamil Digoxin Omeprazole Lansoprazole Candesartan cilexetil Telmisartan Allopurinol Theophylline Indomethacin

Ringer Solution

Serum Ultrafiltrate

100 1.57 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03

100 1.68 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03 ,0.03

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Ther Drug Monit  Volume 37, Number 5, October 2015

patients. Therefore, it is necessary to examine whether this ELISA works in a clinical setting for practical applications.

Dabigatran Enzyme-Linked Immunosorbent Assay

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9. Connolly SJ, Ezekowitz MD, Yusuf S, et al. Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med. 2009;361:1139–1151. 10. Ortel TL. Perioperative management of patients on chronic antithrombotic therapy. Blood. 2012;120:4699–4705. 11. van Ryn J, Stangier J, Haertter S, et al. Dabigatran etexilate–a novel, reversible, oral direct thrombin inhibitor: Interpretation of coagulation assays and reversal of anticoagulant activity. Thromb Haemost. 2010; 103:1116–1127. 12. Shan S, Tanaka H, Shoyama Y. Enzyme-linked immunosorbent assay for glycyrrhizin using anti-glycyrrhizin monoclonal antibody and an eastern blotting technique for glucuronides of glycyrrhetic acid. Anal Chem. 2001;73:5784–5790. 13. Morinaga O, Nakajima S, Tanaka H, et al. Production of monoclonal antibodies against a major purgative component, sennoside B, their characterization and use in ELISA. Analyst. 2001;126:1372–1376. 14. Saita T, Fujito H, Nakano Y, et al. Establishment of an enzyme-linked immunosorbent assay for measurement of sotalol. Biol Pharm Bull. 2004;27:94–96. 15. Saita T, Fujito H, Mori M. A specific and sensitive assay for gefitinib using the enzyme-linked immunosorbent assay in human serum. Biol Pharm Bull. 2005;28:1833–1837. 16. Li XW, Morinaga O, Tian M, et al. Development of an eastern blotting technique for the visual detection of aristolochic acids in aristolochia and asarum species by using a monoclonal antibody against aristolochic acids I and II. Phytochem Anal. 2013;24:645–653. 17. Galfre G, Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 1981;73:3–46. 18. Goding JW. Antibody production by hybridomas. J Immunol Methods. 1980;39:285–308. 19. Weiler EW, Zenk MH, Radioimmunoassay for the determination of digoxin and related compounds in digitalis lanata. Phytochemistry. 1976;15:1537–1545. 20. Goto Y, Shima Y, Morimoto S, et al. Determination of tetrahydrocannabinolic acid—carrier protein conjugate by matrix-assisted laser desorption/ionization mass spectrometry and antibody formation. Org Mass Spectrom. 1994;29:668–671. 21. Shoyama Y, Sakata R, Isobe R, et al. Direct determination of forskolin– bovine serum albumin conjugate by matrix-assisted laser desorption ionization mass spectrometry. Org Mass Spectrom. 1993;28:987–988. 22. Shoyama Y, Fukada T, Tanaka T, et al. Direct determination of opium alkaloid-bovine serum albumin conjugate by matrix-assisted laser desorption/ionization mass spectrometry. Biol Pharm Bull. 1993;16: 1051–1053. 23. Tanaka H, Shoyama Y. Formation of a monoclonal antibody against glycyrrhizin and development of an ELISA. Biol Pharm Bull. 1998;21: 1391–1393. 24. Sakata R, Shoyama Y, Murakami H. Production of monoclonal antibodies and enzyme immunoassay for typical adenylate cyclase activator, forskolin. Cytotechnology. 1994;16:101–108.

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CONCLUSIONS In this study, we succeeded in producing DT-mAb by immunizing mice with a DT-KLH conjugate and used this antibody to develop an ELISA for DT quantitation. Analysis of samples spiked in serum, serum ultrafiltrate, and Ringer solution indicated that this competitive ELISA could be used to monitor the concentration of free DT in blood. The ELISA variability was satisfactory, and the DT-mAb showed a high specificity for DT. This ELISA may provide a highly sensitive and specific tool for DT-TDM and pharmacokinetic studies. ACKNOWLEDGMENTS The authors thank Dr. Hiroyuki Tanaka (Graduate School of Pharmaceutical Sciences, Kyushu University) for the help with MALDI-TOF-MS analysis. REFERENCES

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Generation of an anti-Dabigatran Monoclonal Antibody and Its Use in a Highly Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Serum Dabigatran.

Dabigatran (DT) is a direct thrombin inhibitor used to prevent venous and arterial thromboembolism due to atrial fibrillation. DT is the active form o...
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