Original Papers
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Authors
Supattra Limsuwanchote 1, Juraithip Wungsintaweekul 1, Gorawit Yusakul 3, Jing-Yan Han 2, Kaori Sasaki-Tabata 3, Hiroyuki Tanaka 3, Yukihiro Shoyama 4, Satoshi Morimoto 3
Affiliations
1 2 3 4
Key words " notoginsenoside R1 l " ELISA l " monoclonal antibody l " Panax notoginseng l " Araliaceae l " Sanqi ginseng l
Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla, Thailand Tasly Microcirculation Research Center, Peking University Health Science Center, Beijing, China Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan Department of Pharmacognosy, Nagasaki International University, Sasebo, Nagasaki, Japan
Abstract !
We have prepared a monoclonal antibody against notoginsenoside R1, a primary active constituent of Sanqi ginseng (roots of Panax notoginseng). The monoclonal antibody was raised by immunizing BALB/c male mice with notoginsenoside R1-bovine albumin conjugates following cell fusion via electroporation. This method has been shown to be very effective for producing hybridomas with excellent antibody prevalence following cell fusion of their splenocytes with cells of the myeloma cell line SP2/0. Of all the hybridomas secreting a monoclonal antibody against notoginsenoside
Introduction !
received revised accepted
August 28, 2013 January 7, 2014 January 20, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1360394 Planta Med 2014; 80: 337–342 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence H. Tanaka Department of Pharmacognosy Graduate School of Pharmaceutical Sciences Kyushu University 3–1–1 Maidashi, Higashi-ku Fukuoka 812 8582 Japan Phone: + 81 9 26 42 65 82 Fax: + 81 9 26 42 65 82 [email protected]
The roots of Panax notoginseng (Burk.) F. H. Chen (Araliaceae), also known as Sanqi ginseng, have been used as a traditional medicine in the Yunnan and Guang-Xi provinces in China. In recent years, Sanqi ginseng has attracted significant attention from other countries due to its valuable physiological functions, such as improving blood circulation, ameliorating pathological hemostasis, and relieving pain [1–3]. P. notoginseng contains characteristic saponins called ginsenosides. Of the more than 50 ginsenosides, Rb1, Rg1, and notoginsenoside R1 (NG‑R1) are the major active com" Fig. 1) [4, 5]. These ponents of P. notoginseng (l saponins also have physiological effects, and anti-inflammatory, antioxidative, and central nervous system stimulatory effects, amongst others [6], which greatly contributes to the quality and value of Sanqi ginseng. The rising interest in the health benefits of Sanqi ginseng has brought many products to market, and its sale has increased with each passing year. Therefore, the quality of Sanqi ginseng products must be scientifically evaluated. NG‑R1, a distinctive ginsenoside that shows various pharmaco-
R1, only the 1A1P cell line produces a highly specific antibody to this compound. Surprisingly, the cross-reactivity of this monoclonal antibody for ginsenoside Rg1 and ginsenoside Re, derivatives of protopanaxatriol, was below 1.02 % in a competitive immunoassay. Based on this characteristic of the monoclonal antibody, an indirect competitive ELISA (range of measurement 0.56–9 µg/ mL) was established and applied as a quality control method. In conclusion, the developed immunoassay was easy to handle and reliable for the analysis of notoginsenoside R1 in Sanqi ginseng products without requiring a pretreatment.
logical actions, is a suitable chemical index of Sanqi ginseng quality. Generally, the natural product content in crude drugs is affected by a number of factors, such as the location, and time of growth and harvesting. NG‑R1 is no exception [7], and each Sanqi ginseng sample of ginsenosides varies and must be precisely analyzed to guarantee the quality. Several analytical methods for NG‑R1, such as liquid chromatography with quadrupole time-offlight mass spectrometry (Q‑TOF‑MS) or evaporative light scattering detection, have been reported [8–10]. Also, immunoassays to analyze ginsenosides have been established and applied to the quality control of various ginsengs. We have also prepared anti-ginsenoside Rb1 (G‑Rb1), Rg1 (G‑Rg1), and Re (G‑Re) monoclonal antibodies (mAbs) and successfully implemented ELISA, an immunochromatographic analysis, and Eastern blotting techniques [11–20]. Both anti-G‑Rb1 and G‑Rg1 mAbs were specifically reactive to each ginsenoside [11, 12]. The anti-G‑Re mAb has a high degree of cross-reactivity with G‑Rg1, G‑Rd, and NG‑R1 to roughly the same degree [20]. The present mAbs are not useful for analyzing NG‑R1 concentrations, and there are no anti-
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Preparation of a Monoclonal Antibody against Notoginsenoside R1, a Distinctive Saponin from Panax notoginseng, and Its Application to Indirect Competitive ELISA
Original Papers
Fig. 1 Structure of NG‑R1 and other ginsenosides found in Sanqi ginseng.
Fig. 2 Direct determination of the hapten number of the NG‑R1-BSA conjugates via MALDI‑TOF‑MS.
bodies for NG‑R1 on the market. Accordingly, the purpose of this study was to prepare mAb against NG‑R1 and apply them to an indirect competitive ELISA (icELISA), which could be applicable for the quality control of Sanqi ginseng.
Results and Discussion ! " Fig. 1) suitable for forming a chemiNG‑R1 has sugar moieties (l cal bond to bovine serum albumin (BSA), which provides immunogenicity. During the preparation of the NG‑R1-BSA conjugate immmunogen, NG‑R1 was rapidly converted to an aldehyde via
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periodate oxidation and then conjugated with the lysine or arginine residues of BSA under alkaline conditions. To validate the quality of the prepared immunogen, the hapten number of the NG‑R1-BSA conjugate was determined via a direct analysis of its " Fig. 2. A molecular weight by MALDI‑TOF‑MS, as shown in l broad peak of NG‑R1-BSA in the MALDI‑TOF‑MS spectrum was observed, which means the immunogen sample contained BSA conjugates with different hapten numbers. Judging from the peak at approximately m/z 77 515 and the molecular weights of BSA and NG‑R1 (66 433 and 932, respectively), an average of 11 NG‑R1 molecules were conjugated to the BSA for this particular conjugate. MALDI‑TOF‑MS analysis indicated that the conjugates
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had a sufficient number of haptens and could express the immunogenicity required to raise anti-NG‑R1 antibodies. Additionally, the broad NG‑R1-human serum albumin (HSA) peak, which was used as a solid-phase antigen in the ELISA, also appeared in the MALDI‑TOF‑MS spectrum, and its calculated average hapten number was ten molecules, similar to NG‑R1-BSA. The NG‑R1-BSA conjugate was immunized into a male BALB/c mouse until a specific antibody titer was observed in the blood. After eight immunizations, a sufficient titer was obtained based on indirect ELISA with NG‑R1, and icELISA for free NG‑R1. Antisera produced by the NG‑R1-BSA showed a relatively high specificity for free NG‑R1. The spleen was extirpated from the immunized mouse, and the splenocytes were prepared for cell fusion. Three general methods were established to generate the hybridoma cells: the polyethylene glycol (PEG) method, a method using an envelope of the hemagglutinating virus of Japan (HVJ), and the electroporation (EP) method. We compared the PEG method with the EP method by the number of wells that gave a positive screening result for the ELISAs. As a result, the EP method efficiency was approximately twice that of the PEG method (27.73 % vs. 13.63 %). In total, 188 wells of hybridoma-secreting antiNG‑R1 mAbs were obtained before a second screening involving the indirect competitive ELISA evaluated the characteristics of each hybridoma, specifically for their recognition of each se" Fig. 3 shows the characterization of creted mAb for NG‑R1. l mAbs concerning their cross-reactivities (CRs). Interestingly, the mAb referred to as 1A1P showed a high specificity for NG‑R1 with no CR for other ginsenosides. This hybridoma cell line, which produced an mAb reactive to NG‑R1, was cloned via a limiting dilution method, and the mAb was classified by the IgG1 category, which had κ light chains. Anti-NG‑R1 mAbs were purified from the hybridoma cell culture supernatants and applied to an ELISA, which is the most common and practical immunoassay. Because NG‑R1 is classified as a hapten and cannot be immobilized on an appropriate support material, a competitive format was applied to the immunoassays for detecting NG‑R1. To improve the icELISA sensitivity, the antiNG‑R1 mAb concentration must be as low as possible to allow for the proper chromogenic development in a directive ELISA coated with the NG‑R1-HSA conjugate. The result of the directive ELISA for a variety of anti-NG‑R1 mAbs indicated the appropriate antiNG‑R1 mAb concentration for icELISA was 7.4 µg/mL, which gave an absorbance at 405 nm of approximately 0.8–1.0. " Fig. 4 shows a calibration curve for detecting NG‑R1 in icELISA l based on these conditions. When the first antibody was applied, a
Fig. 4 Calibration curves for NG‑R1 in icELISA using the anti-NG‑R1 mAb 1A1P. The mAb 1A1P and NG‑R1–HSA coating conjugate concentrations were 7.4 and 1 µg/mL, respectively.
twofold serial dilution of NG‑R1 was applied to the micro-immunoplate, and it competed with the binding of the anti-NG‑R1 mAb 1A1P to NG‑R1-HSA. After treatment with a secondary antibody and adding a substrate to the plate, the development of a color gradation was distinguished for increasing NG‑R1 concentrations, which produces a standard curve. For this established system, the measuring range of NG‑R1 was from 0.563 to 9 µg/mL. The specificity of the anti-NG‑R1 mAb 1A1P was determined by the cross-reactivity of various ginsenosides using the icELISA and was calculated using the equation developed by Weiler and " Table 1, only NG‑R1 was recognized, Zenk [21]. As shown in l and the CR for G‑Rb1 was 2.61 %, whereas the four ginsenosides and other natural products had CR values below 1.02 %. These experimental data suggest that the anti-NG‑R1 mAb 1A1P only reacts with NG‑R1, which is a promising characteristic for the determination of NG‑R1 in Sanqi ginseng. " Table 2 shows the result of the intra-assay and inter-assay prel cision tests for icELISA using anti-NG‑R1 mAb. The intra-assay precision was evaluated based on the variation and repeatability of the NG‑R1 test for wells on the same plate (n = 4), whereas the inter-assay precision was compared between different plates (n = 4). The maximum relative standard deviation (RSD) of the intra-assay was 4.16 %, while that of the inter-assay was 7.73 %. A recovery experiment was conducted to confirm the reliability " Table 3 shows that good of the icELISA procedure developed. l Limsuwanchote S et al. Preparation of a …
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Fig. 3 CRs for various mAbs for ginsenosides during the icELISA. The NG‑R1–OVA coated immunoplate (1 µg/mL, 100 µL) was treated with a supernatant from each hybridoma clone and 200 µg/mL of ginsenoside solution. The inhibition was calculated as (1 – A)/A0 × 100 (%), where A is the absorbance in the presence of the ginsenoside, and A0 is the absorbance in the absence of the test compound (20 % MeOH solution).
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CR (%)a
Compound Notoginsenoside R1 Ginsenoside Rb1 Ginsenoside Rg1 Ginsenoside Re Ginsenoside Rc Ginsenoside Rd Glycyrrhizin Digitonin Saikosaponin a Saikosaponin d Cholesterol Ergosterol β-Sitosterol Ursolic acid Deoxycholic acid Swertiamarin Quercetin Rutin a
100 2.61 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02
a
NG‑R1
Intra-assay
Inter-assay
(µg/mL)
RSDa (%, n = 4)
RSD (%, n = 4)
8 6 4 2 1
1.37 0.59 3.54 4.16 2.42
2.26 1.74 7.73 2.63 4.80
RSD: relative standard deviation
ta would be valuable information in the determination of the quality of Sanqi ginseng materials. In this study, a novel mAb 1A1P for NG‑R1 from Sanqi ginseng was prepared and characterized. Interestingly, this mAb only recognized free NG‑R1 during the icELISA. This novel characteristic potentially enables various applications in the pharmaceutical and food sciences. First of all, we used the mAb to establish an icELISA and confirm the reliability of the system as a scientific quality control method for Sanqi ginseng with high sensitivity. The icELISA requires no cumbersome sample pretreatment, and many samples can be analyzed over a short period.
CR were calculated using the equation given in Materials and Methods [21]
recoveries of NG‑R1 from Sanqi ginseng sample solutions were observed in this experiment, which ranged from 99.5 % to 104 % with an RSD of 2.09–7.59 %. The recovery of NG‑R1 from each spiked sample was almost 100 %, which demonstrated the high reliability of this immunoassay. To confirm the icELISA for the quantitative analysis of NG‑R1 in Sanqi ginseng, five samples were purchased from a market in Japan. A brief sample preparation was adopted using a very small amount (25 mg) of Sanqi ginseng powder. A methanol extract was then prepared and diluted for the analysis of NG‑R1 in the " Table 4 shows the quantitative data for the icELISA desamples. l termination using the anti-NG‑R1 mAb 1A1P. Contrary to expectations, the results are not in accordance with those previously reported [9]. The calculated NG‑R1 content of the Sanqi ginseng was approximately ten times the normal amount. These results obviously depend on the CRs of the mAb against other ginsenosides, and we have no means of evaluating its specificity. The unintended consequence of this quantitative analysis of NG‑R1 using the icELISA is a formidable challenge for clarifying what compounds are reactive to the mAb. We plan to adopt an immunoaffinity-based approach to identify these cross-reactive substances in the future. This experiment could provide useful information for interpreting previous data from the icELISA with the antiNG‑R1 mAb. At present, it is supposed that the quantitative data provided by the icELISA using the anti-NG‑R1 mAb 1A1P must reflect productivity of the ginsenosides in P. notoginseng. These da-
a
Table 2 Intra- and inter-assay precision of NG‑R1 analysis using icELISA.
Materials and Methods !
Chemicals and reagents NG‑R1 (purity, > 98%) was purchased from Carbosynth Limited, whereas ginsenosides Re, Rg1, Rb1, Rc, and Rd (purity > 98 %) were purchased from Wako Pure Chemical Industries, Ltd. BSA and HSA were obtained from Sigma-Aldrich. Peroxidase-labeled anti-mouse IgG was provided by MP Biomedicals, LLC. Freundʼs complete and incomplete adjuvants were procured from DIFCO Laboratories. All other chemicals were of analytical reagent grade.
Sample preparation Different ginseng samples (25 mg) were extracted with MeOH (1 mL) by sonicating for 30 min. The extracts were centrifuged at 10 000 rpm for 2 min. After repeating this extraction procedure five times, the supernatants were combined and evaporated to dryness. The residues were reconstituted to 1 mL with MeOH and diluted to the appropriate concentration for ELISA using a 20 % MeOH solution.
Synthesis of antigen conjugates NG‑R1 was conjugated to carrier proteins via periodate oxidation [22]. NG‑R1 (10 mg) was dissolved in an 80% MeOH solution (0.7 mL) and added dropwise to 0.5 mL of an NaIO4 solution
Spiked NG‑R1 concentration
Measured concentrationa
RSDb
Recovery
(µg/mL)
(µg/mL)
(%)
(%)
0 25.0 50.0 75.0 100 200
211 237 261 285 314 413
2.09 3.96 5.81 7.59 5.79 5.20
– 104 100 99.5 104 101
Data are the mean based on triplicate analyses of each sample. b RSD: relative standard deviation
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Table 3 Recovery experiment of spiked NG‑R1 in Sanqi ginseng samples using icELISA with the anti-NG‑R1 mAb 1A1P.
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Table 1 CRs of anti-NG‑R1 mAb 1A1P with NG‑R1 and structurally related compounds.
Table 4 Determination of NG‑R1 in Sanqi ginseng samples using icELISA with the anti-NG‑R1 mAb 1A1P.
a
Sample
NG‑R1 contenta (µg/mg dry wt.)
Sanqi ginseng 1 Sanqi ginseng 2 Sanqi ginseng 3 Sanqi ginseng 4 Sanqi ginseng 5
70.5 ± 5.1 51.5 ± 1.8 43.7 ± 2.6 57.1 ± 3.7 69.9 ± 0.7
Data are the mean ± SD based on triplicate analyses of each sample
(4 mg/0.5 mL). The reaction mixture was stirred at room temperature for 1 h. A carbonate buffer (50 mM, pH 9.6, 1 mL) containing BSA or HSA (6 mg) was added to the above solution and stirred at room temperature for 5 h. Subsequently, the solution was dialyzed against H2O five times at 4 °C and then lyophilized to yield 5.8 mg of NG‑R1-BSA conjugate. The NG‑R1-HSA conjugate (6.7 mg) was obtained in the same manner.
Determination of the hapten number of the antigen conjugates using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry The hapten number of the NG‑R1 conjugates was determined via MALDI‑TOF mass spectrometry as previously described [23]. Briefly, a small amount (1–10 pmol) of the NG‑R1 conjugate was mixed with a 103-fold molar excess of sinapinic acid in a 0.15 % trifluoroacetic acid solution. The mixture was deposited on the target plate (Bruker Daltonics) and allowed to air dry. The sample was injected into a MALDI‑TOF‑MS system (Bruker AutoFlex III), and the data were analyzed using flexAnalysis 3.0.92 software.
Animal treatment BALB/c mice were purchased from KBT Oriental Co. A standard diet (MF; Oriental Yeast Co.) and water were provided ad libitum. The research procedures and animal care were approved by the Committee on Ethics of Animal Experiments, Graduate School of Pharmaceutical Sciences, Kyushu University, Japan, and were conducted according to the Guidelines for Animal Experiments of the Graduate School of Pharmaceutical Sciences, Kyushu University (Animal ethic reference no. A22-113-0 dated March 10, 2010, Kyushu University, Japan).
Immunization, hybridization, and monoclonal antibody production BALB/c male mice, 5 weeks old, were immunized intraperitoneally with a 1 : 1 emulsion of NG‑R1-BSA (100 µg/0.1 mL) in Freundʼs complete adjuvant. A second immunization was injected using a 1 : 1 emulsion of NG‑R1-BSA (100 µg/0.5 mL protein) in Freundʼs incomplete adjuvant two weeks after the first immunization. A third immunization was performed by injecting NG‑R1-BSA (100 µg/0.5 mL protein) in phosphate buffer saline (PBS) two weeks after the second immunization. The same amounts (100 µg/0.5 mL protein) of NG‑R1-BSA in PBS were injected into the mice five more times at the same interval of two weeks. On the third day after the final immunization, the mice were sacrificed and the splenocytes were isolated. These splenocytes were fused with a HAT-sensitive myeloma cell, SP2/0, via PEG [24] and electrofusion methods [25]. PEG 1500 was fused with 5.7 × 107 SP2/0 cells and 5.7 × 108 spleen cells according to the standard protocol [11, 12, 22, 26]. For electrofusion, 1.9 × 107 SP2/0 cells and 1.9 × 108 spleen cells
were mixed and treated with an EP buffer containing 0.28 M of mannitol, 0.1 mM of CaCl2, and 0.1 mM of MgCl2. The cell suspensions were centrifuged at 1200 rpm for 5 min and resuspended in 10 mL of the EP buffer. The total number of cells was adjusted to 1–5 × 107 cells/mL using the EP buffer before being added to the fusion chamber. Electrofusion was performed in a microchamber (2-mm gap) using platinum electrodes (CUY497P2). An electric field pulse was generated using an Electro-Cell Fusion Generator (LF 201; Nepa Gene Co. Ltd.). An alternating electric field was applied at 30 V/cm for 20 s followed by three electric pulses at 350 V/cm for 30 µs at an interval of 0.5 s to break down the cell membranes. The alternating electric field was then reapplied for another 7 s. The fused cells were transferred to an incubator at 37 °C for 20 min. Next, 8 mL of enriched RPMI 1640–DulbeccoʼsHams F12 (eRDF) medium containing 20 % fetal bovine serum (FBS) was added to the fused cells, which were then centrifuged at 1200 rpm for 5 min. The cell pellets were suspended in the media used above and seeded into a 96-well tissue culture plate. The hybridoma supernatants were screened for the presence of anti-NG‑R1 antibodies using an indirect ELISA. Those hybridomas that produced mAbs against NG‑R1 were cloned via the limiting dilution method [27] and cultured in an eRDF medium using 10 % FBS. Finally, the established hybridomas were cultured on a large scale using eRDF supplemented with RD-1 (5 µg/mL insulin, 10 µg/mL transferrin, 25 µM ethanolamine, and 25 nM sodium selenite) [28].
Purification of the monoclonal antibodies Anti-NG‑R1 mAb was purified using an immobilized protein A column. A cultured medium (500 mL) containing IgG was loaded onto the column and washed with 10 mM phosphate buffer (pH 7). The bound IgG was eluted with 100 mM citrate buffer (pH 2.7) and neutralized with 1 M Tris-HCl buffer (pH 9.0). The purified mAb solution was concentrated in an ultra-centrifuge tube (Millipore) to obtain the concentrated mAb.
Indirect ELISA using notoginsenoside R1-human serum albumin The NG‑R1-HSA conjugate was dissolved in a 50-mM carbonate buffer (pH 9.6) (100 µL, 1 µg/mL) and adsorbed to the wells of a 96-well plate. The wells were then blocked with 300 µL of 5 % skim milk in PBS (SPBS) for 1 h to reduced nonspecific adsorption. The plate was washed three times with 0.05 % Tween 20 in phosphate buffered saline (TPBS) and reacted for 1 h with either the hybridoma supernatants or a test mAb (100 µL). After this washing step, the bound antibodies were incubated for 1 h with 100 µL of a 1 : 1000 dilution of the peroxidase-anti-mouse IgG. After washing three times with TPBS, 100 µL of a substrate solution [0.1 M citrate buffer (pH 4.0) containing 0.003 % H2O2 and 0.3 mg/mL of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (Wako Pure Chemical Ind., Ltd.)] was added and incubated for 15 min. The absorbance at 405 nm was measured using a microplate reader (Multiskan FC microplate photometer; Thermo Scientific). All reactions were performed at 37 °C.
Indirect competitive ELISA using anti-notoginseng-R1 monoclonal antibody An indirect competitive ELISA was performed as described above with slight modifications. After blocking the plate with SPBS and washing three times, 50 µL of NG-R1 or the ginseng samples at varying concentrations in 20 % MeOH were incubated for 1 hr
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with 50 µL of the mAb solution (7.4 µg/mL). The plate was washed three times with TPBS and combined with 100 µL of a 1 : 1000 dilution of peroxidase-anti-mouse IgG for 1 hr. Subsequently, 100 µL of the substrate solution was added to each well and incubated for 15 min. The absorbance was read at 405 nm. The CR of the NG‑R1 and related compounds was calculated using Weiler and Zenkʼs equation [21]: % Cross reactivity (CR) = [(concentration of NG‑R1 yielding A/ A0 = 50 %)/ (concentration of the related compound yielding A/A0 = 50 %)] × 100 where A is the absorbance in the presence of the test compound, and A0 is the absorbance in the absence of the test compound (20% MeOH solution).
Recovery of notoginseng-R1 from ginseng samples A Sanqi ginseng sample (36 mg) was extracted five times with 18 mL MeOH at a time using sonication. All extracts were combined and divided into 18 samples. Each ginseng extract sample (1 mL) was spiked with different amounts of a standard solution of NG‑R1: 25, 50, 75, 100, and 200 µg (n = 3). Each extract was evaporated to dryness and then dissolved in 1 mL MeOH. All samples were analyzed by the described ELISA method. For unspiked samples, a concentration of 211 µg/mL NG‑R1 was determined. The recovery (%) for spiked samples was calculated on the basis of the amount of measured and spiked NG‑R1: Recoveryð%Þ ¼
Measured amount of NR R1 211 100 Spiked amount of NG R1
Acknowledgments !
This study was supported, in part, by a Grant-in-Aid from JSPSʼs Asian CORE Program, the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the research fund of the Kyushu University Foundation and the Royal Golden Jubilee Ph. D Program, and the Thailand Research Fund (PHD/0311/2550).
Conflict of Interest !
The authors have declared no competing interests (financial or otherwise) related to this manuscript.
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Authors
Supattra Limsuwanchote 1, Juraithip Wungsintaweekul 1, Gorawit Yusakul 3, Jing-Yan Han 2, Kaori Sasaki-Tabata 3, Hiroyuki Tanaka 3, Yukihiro Shoyama 4, Satoshi Morimoto 3
Affiliations
1 2 3 4
Key words " notoginsenoside R1 l " ELISA l " monoclonal antibody l " Panax notoginseng l " Araliaceae l " Sanqi ginseng l
Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla, Thailand Tasly Microcirculation Research Center, Peking University Health Science Center, Beijing, China Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan Department of Pharmacognosy, Nagasaki International University, Sasebo, Nagasaki, Japan
Abstract !
We have prepared a monoclonal antibody against notoginsenoside R1, a primary active constituent of Sanqi ginseng (roots of Panax notoginseng). The monoclonal antibody was raised by immunizing BALB/c male mice with notoginsenoside R1-bovine albumin conjugates following cell fusion via electroporation. This method has been shown to be very effective for producing hybridomas with excellent antibody prevalence following cell fusion of their splenocytes with cells of the myeloma cell line SP2/0. Of all the hybridomas secreting a monoclonal antibody against notoginsenoside
Introduction !
received revised accepted
August 28, 2013 January 7, 2014 January 20, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1360394 Planta Med 2014; 80: 337–342 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence H. Tanaka Department of Pharmacognosy Graduate School of Pharmaceutical Sciences Kyushu University 3–1–1 Maidashi, Higashi-ku Fukuoka 812 8582 Japan Phone: + 81 9 26 42 65 82 Fax: + 81 9 26 42 65 82 [email protected]
The roots of Panax notoginseng (Burk.) F. H. Chen (Araliaceae), also known as Sanqi ginseng, have been used as a traditional medicine in the Yunnan and Guang-Xi provinces in China. In recent years, Sanqi ginseng has attracted significant attention from other countries due to its valuable physiological functions, such as improving blood circulation, ameliorating pathological hemostasis, and relieving pain [1–3]. P. notoginseng contains characteristic saponins called ginsenosides. Of the more than 50 ginsenosides, Rb1, Rg1, and notoginsenoside R1 (NG‑R1) are the major active com" Fig. 1) [4, 5]. These ponents of P. notoginseng (l saponins also have physiological effects, and anti-inflammatory, antioxidative, and central nervous system stimulatory effects, amongst others [6], which greatly contributes to the quality and value of Sanqi ginseng. The rising interest in the health benefits of Sanqi ginseng has brought many products to market, and its sale has increased with each passing year. Therefore, the quality of Sanqi ginseng products must be scientifically evaluated. NG‑R1, a distinctive ginsenoside that shows various pharmaco-
R1, only the 1A1P cell line produces a highly specific antibody to this compound. Surprisingly, the cross-reactivity of this monoclonal antibody for ginsenoside Rg1 and ginsenoside Re, derivatives of protopanaxatriol, was below 1.02 % in a competitive immunoassay. Based on this characteristic of the monoclonal antibody, an indirect competitive ELISA (range of measurement 0.56–9 µg/ mL) was established and applied as a quality control method. In conclusion, the developed immunoassay was easy to handle and reliable for the analysis of notoginsenoside R1 in Sanqi ginseng products without requiring a pretreatment.
logical actions, is a suitable chemical index of Sanqi ginseng quality. Generally, the natural product content in crude drugs is affected by a number of factors, such as the location, and time of growth and harvesting. NG‑R1 is no exception [7], and each Sanqi ginseng sample of ginsenosides varies and must be precisely analyzed to guarantee the quality. Several analytical methods for NG‑R1, such as liquid chromatography with quadrupole time-offlight mass spectrometry (Q‑TOF‑MS) or evaporative light scattering detection, have been reported [8–10]. Also, immunoassays to analyze ginsenosides have been established and applied to the quality control of various ginsengs. We have also prepared anti-ginsenoside Rb1 (G‑Rb1), Rg1 (G‑Rg1), and Re (G‑Re) monoclonal antibodies (mAbs) and successfully implemented ELISA, an immunochromatographic analysis, and Eastern blotting techniques [11–20]. Both anti-G‑Rb1 and G‑Rg1 mAbs were specifically reactive to each ginsenoside [11, 12]. The anti-G‑Re mAb has a high degree of cross-reactivity with G‑Rg1, G‑Rd, and NG‑R1 to roughly the same degree [20]. The present mAbs are not useful for analyzing NG‑R1 concentrations, and there are no anti-
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Preparation of a Monoclonal Antibody against Notoginsenoside R1, a Distinctive Saponin from Panax notoginseng, and Its Application to Indirect Competitive ELISA
Original Papers
Fig. 1 Structure of NG‑R1 and other ginsenosides found in Sanqi ginseng.
Fig. 2 Direct determination of the hapten number of the NG‑R1-BSA conjugates via MALDI‑TOF‑MS.
bodies for NG‑R1 on the market. Accordingly, the purpose of this study was to prepare mAb against NG‑R1 and apply them to an indirect competitive ELISA (icELISA), which could be applicable for the quality control of Sanqi ginseng.
Results and Discussion ! " Fig. 1) suitable for forming a chemiNG‑R1 has sugar moieties (l cal bond to bovine serum albumin (BSA), which provides immunogenicity. During the preparation of the NG‑R1-BSA conjugate immmunogen, NG‑R1 was rapidly converted to an aldehyde via
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periodate oxidation and then conjugated with the lysine or arginine residues of BSA under alkaline conditions. To validate the quality of the prepared immunogen, the hapten number of the NG‑R1-BSA conjugate was determined via a direct analysis of its " Fig. 2. A molecular weight by MALDI‑TOF‑MS, as shown in l broad peak of NG‑R1-BSA in the MALDI‑TOF‑MS spectrum was observed, which means the immunogen sample contained BSA conjugates with different hapten numbers. Judging from the peak at approximately m/z 77 515 and the molecular weights of BSA and NG‑R1 (66 433 and 932, respectively), an average of 11 NG‑R1 molecules were conjugated to the BSA for this particular conjugate. MALDI‑TOF‑MS analysis indicated that the conjugates
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had a sufficient number of haptens and could express the immunogenicity required to raise anti-NG‑R1 antibodies. Additionally, the broad NG‑R1-human serum albumin (HSA) peak, which was used as a solid-phase antigen in the ELISA, also appeared in the MALDI‑TOF‑MS spectrum, and its calculated average hapten number was ten molecules, similar to NG‑R1-BSA. The NG‑R1-BSA conjugate was immunized into a male BALB/c mouse until a specific antibody titer was observed in the blood. After eight immunizations, a sufficient titer was obtained based on indirect ELISA with NG‑R1, and icELISA for free NG‑R1. Antisera produced by the NG‑R1-BSA showed a relatively high specificity for free NG‑R1. The spleen was extirpated from the immunized mouse, and the splenocytes were prepared for cell fusion. Three general methods were established to generate the hybridoma cells: the polyethylene glycol (PEG) method, a method using an envelope of the hemagglutinating virus of Japan (HVJ), and the electroporation (EP) method. We compared the PEG method with the EP method by the number of wells that gave a positive screening result for the ELISAs. As a result, the EP method efficiency was approximately twice that of the PEG method (27.73 % vs. 13.63 %). In total, 188 wells of hybridoma-secreting antiNG‑R1 mAbs were obtained before a second screening involving the indirect competitive ELISA evaluated the characteristics of each hybridoma, specifically for their recognition of each se" Fig. 3 shows the characterization of creted mAb for NG‑R1. l mAbs concerning their cross-reactivities (CRs). Interestingly, the mAb referred to as 1A1P showed a high specificity for NG‑R1 with no CR for other ginsenosides. This hybridoma cell line, which produced an mAb reactive to NG‑R1, was cloned via a limiting dilution method, and the mAb was classified by the IgG1 category, which had κ light chains. Anti-NG‑R1 mAbs were purified from the hybridoma cell culture supernatants and applied to an ELISA, which is the most common and practical immunoassay. Because NG‑R1 is classified as a hapten and cannot be immobilized on an appropriate support material, a competitive format was applied to the immunoassays for detecting NG‑R1. To improve the icELISA sensitivity, the antiNG‑R1 mAb concentration must be as low as possible to allow for the proper chromogenic development in a directive ELISA coated with the NG‑R1-HSA conjugate. The result of the directive ELISA for a variety of anti-NG‑R1 mAbs indicated the appropriate antiNG‑R1 mAb concentration for icELISA was 7.4 µg/mL, which gave an absorbance at 405 nm of approximately 0.8–1.0. " Fig. 4 shows a calibration curve for detecting NG‑R1 in icELISA l based on these conditions. When the first antibody was applied, a
Fig. 4 Calibration curves for NG‑R1 in icELISA using the anti-NG‑R1 mAb 1A1P. The mAb 1A1P and NG‑R1–HSA coating conjugate concentrations were 7.4 and 1 µg/mL, respectively.
twofold serial dilution of NG‑R1 was applied to the micro-immunoplate, and it competed with the binding of the anti-NG‑R1 mAb 1A1P to NG‑R1-HSA. After treatment with a secondary antibody and adding a substrate to the plate, the development of a color gradation was distinguished for increasing NG‑R1 concentrations, which produces a standard curve. For this established system, the measuring range of NG‑R1 was from 0.563 to 9 µg/mL. The specificity of the anti-NG‑R1 mAb 1A1P was determined by the cross-reactivity of various ginsenosides using the icELISA and was calculated using the equation developed by Weiler and " Table 1, only NG‑R1 was recognized, Zenk [21]. As shown in l and the CR for G‑Rb1 was 2.61 %, whereas the four ginsenosides and other natural products had CR values below 1.02 %. These experimental data suggest that the anti-NG‑R1 mAb 1A1P only reacts with NG‑R1, which is a promising characteristic for the determination of NG‑R1 in Sanqi ginseng. " Table 2 shows the result of the intra-assay and inter-assay prel cision tests for icELISA using anti-NG‑R1 mAb. The intra-assay precision was evaluated based on the variation and repeatability of the NG‑R1 test for wells on the same plate (n = 4), whereas the inter-assay precision was compared between different plates (n = 4). The maximum relative standard deviation (RSD) of the intra-assay was 4.16 %, while that of the inter-assay was 7.73 %. A recovery experiment was conducted to confirm the reliability " Table 3 shows that good of the icELISA procedure developed. l Limsuwanchote S et al. Preparation of a …
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Fig. 3 CRs for various mAbs for ginsenosides during the icELISA. The NG‑R1–OVA coated immunoplate (1 µg/mL, 100 µL) was treated with a supernatant from each hybridoma clone and 200 µg/mL of ginsenoside solution. The inhibition was calculated as (1 – A)/A0 × 100 (%), where A is the absorbance in the presence of the ginsenoside, and A0 is the absorbance in the absence of the test compound (20 % MeOH solution).
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CR (%)a
Compound Notoginsenoside R1 Ginsenoside Rb1 Ginsenoside Rg1 Ginsenoside Re Ginsenoside Rc Ginsenoside Rd Glycyrrhizin Digitonin Saikosaponin a Saikosaponin d Cholesterol Ergosterol β-Sitosterol Ursolic acid Deoxycholic acid Swertiamarin Quercetin Rutin a
100 2.61 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02 < 1.02
a
NG‑R1
Intra-assay
Inter-assay
(µg/mL)
RSDa (%, n = 4)
RSD (%, n = 4)
8 6 4 2 1
1.37 0.59 3.54 4.16 2.42
2.26 1.74 7.73 2.63 4.80
RSD: relative standard deviation
ta would be valuable information in the determination of the quality of Sanqi ginseng materials. In this study, a novel mAb 1A1P for NG‑R1 from Sanqi ginseng was prepared and characterized. Interestingly, this mAb only recognized free NG‑R1 during the icELISA. This novel characteristic potentially enables various applications in the pharmaceutical and food sciences. First of all, we used the mAb to establish an icELISA and confirm the reliability of the system as a scientific quality control method for Sanqi ginseng with high sensitivity. The icELISA requires no cumbersome sample pretreatment, and many samples can be analyzed over a short period.
CR were calculated using the equation given in Materials and Methods [21]
recoveries of NG‑R1 from Sanqi ginseng sample solutions were observed in this experiment, which ranged from 99.5 % to 104 % with an RSD of 2.09–7.59 %. The recovery of NG‑R1 from each spiked sample was almost 100 %, which demonstrated the high reliability of this immunoassay. To confirm the icELISA for the quantitative analysis of NG‑R1 in Sanqi ginseng, five samples were purchased from a market in Japan. A brief sample preparation was adopted using a very small amount (25 mg) of Sanqi ginseng powder. A methanol extract was then prepared and diluted for the analysis of NG‑R1 in the " Table 4 shows the quantitative data for the icELISA desamples. l termination using the anti-NG‑R1 mAb 1A1P. Contrary to expectations, the results are not in accordance with those previously reported [9]. The calculated NG‑R1 content of the Sanqi ginseng was approximately ten times the normal amount. These results obviously depend on the CRs of the mAb against other ginsenosides, and we have no means of evaluating its specificity. The unintended consequence of this quantitative analysis of NG‑R1 using the icELISA is a formidable challenge for clarifying what compounds are reactive to the mAb. We plan to adopt an immunoaffinity-based approach to identify these cross-reactive substances in the future. This experiment could provide useful information for interpreting previous data from the icELISA with the antiNG‑R1 mAb. At present, it is supposed that the quantitative data provided by the icELISA using the anti-NG‑R1 mAb 1A1P must reflect productivity of the ginsenosides in P. notoginseng. These da-
a
Table 2 Intra- and inter-assay precision of NG‑R1 analysis using icELISA.
Materials and Methods !
Chemicals and reagents NG‑R1 (purity, > 98%) was purchased from Carbosynth Limited, whereas ginsenosides Re, Rg1, Rb1, Rc, and Rd (purity > 98 %) were purchased from Wako Pure Chemical Industries, Ltd. BSA and HSA were obtained from Sigma-Aldrich. Peroxidase-labeled anti-mouse IgG was provided by MP Biomedicals, LLC. Freundʼs complete and incomplete adjuvants were procured from DIFCO Laboratories. All other chemicals were of analytical reagent grade.
Sample preparation Different ginseng samples (25 mg) were extracted with MeOH (1 mL) by sonicating for 30 min. The extracts were centrifuged at 10 000 rpm for 2 min. After repeating this extraction procedure five times, the supernatants were combined and evaporated to dryness. The residues were reconstituted to 1 mL with MeOH and diluted to the appropriate concentration for ELISA using a 20 % MeOH solution.
Synthesis of antigen conjugates NG‑R1 was conjugated to carrier proteins via periodate oxidation [22]. NG‑R1 (10 mg) was dissolved in an 80% MeOH solution (0.7 mL) and added dropwise to 0.5 mL of an NaIO4 solution
Spiked NG‑R1 concentration
Measured concentrationa
RSDb
Recovery
(µg/mL)
(µg/mL)
(%)
(%)
0 25.0 50.0 75.0 100 200
211 237 261 285 314 413
2.09 3.96 5.81 7.59 5.79 5.20
– 104 100 99.5 104 101
Data are the mean based on triplicate analyses of each sample. b RSD: relative standard deviation
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Table 3 Recovery experiment of spiked NG‑R1 in Sanqi ginseng samples using icELISA with the anti-NG‑R1 mAb 1A1P.
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Table 1 CRs of anti-NG‑R1 mAb 1A1P with NG‑R1 and structurally related compounds.
Table 4 Determination of NG‑R1 in Sanqi ginseng samples using icELISA with the anti-NG‑R1 mAb 1A1P.
a
Sample
NG‑R1 contenta (µg/mg dry wt.)
Sanqi ginseng 1 Sanqi ginseng 2 Sanqi ginseng 3 Sanqi ginseng 4 Sanqi ginseng 5
70.5 ± 5.1 51.5 ± 1.8 43.7 ± 2.6 57.1 ± 3.7 69.9 ± 0.7
Data are the mean ± SD based on triplicate analyses of each sample
(4 mg/0.5 mL). The reaction mixture was stirred at room temperature for 1 h. A carbonate buffer (50 mM, pH 9.6, 1 mL) containing BSA or HSA (6 mg) was added to the above solution and stirred at room temperature for 5 h. Subsequently, the solution was dialyzed against H2O five times at 4 °C and then lyophilized to yield 5.8 mg of NG‑R1-BSA conjugate. The NG‑R1-HSA conjugate (6.7 mg) was obtained in the same manner.
Determination of the hapten number of the antigen conjugates using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry The hapten number of the NG‑R1 conjugates was determined via MALDI‑TOF mass spectrometry as previously described [23]. Briefly, a small amount (1–10 pmol) of the NG‑R1 conjugate was mixed with a 103-fold molar excess of sinapinic acid in a 0.15 % trifluoroacetic acid solution. The mixture was deposited on the target plate (Bruker Daltonics) and allowed to air dry. The sample was injected into a MALDI‑TOF‑MS system (Bruker AutoFlex III), and the data were analyzed using flexAnalysis 3.0.92 software.
Animal treatment BALB/c mice were purchased from KBT Oriental Co. A standard diet (MF; Oriental Yeast Co.) and water were provided ad libitum. The research procedures and animal care were approved by the Committee on Ethics of Animal Experiments, Graduate School of Pharmaceutical Sciences, Kyushu University, Japan, and were conducted according to the Guidelines for Animal Experiments of the Graduate School of Pharmaceutical Sciences, Kyushu University (Animal ethic reference no. A22-113-0 dated March 10, 2010, Kyushu University, Japan).
Immunization, hybridization, and monoclonal antibody production BALB/c male mice, 5 weeks old, were immunized intraperitoneally with a 1 : 1 emulsion of NG‑R1-BSA (100 µg/0.1 mL) in Freundʼs complete adjuvant. A second immunization was injected using a 1 : 1 emulsion of NG‑R1-BSA (100 µg/0.5 mL protein) in Freundʼs incomplete adjuvant two weeks after the first immunization. A third immunization was performed by injecting NG‑R1-BSA (100 µg/0.5 mL protein) in phosphate buffer saline (PBS) two weeks after the second immunization. The same amounts (100 µg/0.5 mL protein) of NG‑R1-BSA in PBS were injected into the mice five more times at the same interval of two weeks. On the third day after the final immunization, the mice were sacrificed and the splenocytes were isolated. These splenocytes were fused with a HAT-sensitive myeloma cell, SP2/0, via PEG [24] and electrofusion methods [25]. PEG 1500 was fused with 5.7 × 107 SP2/0 cells and 5.7 × 108 spleen cells according to the standard protocol [11, 12, 22, 26]. For electrofusion, 1.9 × 107 SP2/0 cells and 1.9 × 108 spleen cells
were mixed and treated with an EP buffer containing 0.28 M of mannitol, 0.1 mM of CaCl2, and 0.1 mM of MgCl2. The cell suspensions were centrifuged at 1200 rpm for 5 min and resuspended in 10 mL of the EP buffer. The total number of cells was adjusted to 1–5 × 107 cells/mL using the EP buffer before being added to the fusion chamber. Electrofusion was performed in a microchamber (2-mm gap) using platinum electrodes (CUY497P2). An electric field pulse was generated using an Electro-Cell Fusion Generator (LF 201; Nepa Gene Co. Ltd.). An alternating electric field was applied at 30 V/cm for 20 s followed by three electric pulses at 350 V/cm for 30 µs at an interval of 0.5 s to break down the cell membranes. The alternating electric field was then reapplied for another 7 s. The fused cells were transferred to an incubator at 37 °C for 20 min. Next, 8 mL of enriched RPMI 1640–DulbeccoʼsHams F12 (eRDF) medium containing 20 % fetal bovine serum (FBS) was added to the fused cells, which were then centrifuged at 1200 rpm for 5 min. The cell pellets were suspended in the media used above and seeded into a 96-well tissue culture plate. The hybridoma supernatants were screened for the presence of anti-NG‑R1 antibodies using an indirect ELISA. Those hybridomas that produced mAbs against NG‑R1 were cloned via the limiting dilution method [27] and cultured in an eRDF medium using 10 % FBS. Finally, the established hybridomas were cultured on a large scale using eRDF supplemented with RD-1 (5 µg/mL insulin, 10 µg/mL transferrin, 25 µM ethanolamine, and 25 nM sodium selenite) [28].
Purification of the monoclonal antibodies Anti-NG‑R1 mAb was purified using an immobilized protein A column. A cultured medium (500 mL) containing IgG was loaded onto the column and washed with 10 mM phosphate buffer (pH 7). The bound IgG was eluted with 100 mM citrate buffer (pH 2.7) and neutralized with 1 M Tris-HCl buffer (pH 9.0). The purified mAb solution was concentrated in an ultra-centrifuge tube (Millipore) to obtain the concentrated mAb.
Indirect ELISA using notoginsenoside R1-human serum albumin The NG‑R1-HSA conjugate was dissolved in a 50-mM carbonate buffer (pH 9.6) (100 µL, 1 µg/mL) and adsorbed to the wells of a 96-well plate. The wells were then blocked with 300 µL of 5 % skim milk in PBS (SPBS) for 1 h to reduced nonspecific adsorption. The plate was washed three times with 0.05 % Tween 20 in phosphate buffered saline (TPBS) and reacted for 1 h with either the hybridoma supernatants or a test mAb (100 µL). After this washing step, the bound antibodies were incubated for 1 h with 100 µL of a 1 : 1000 dilution of the peroxidase-anti-mouse IgG. After washing three times with TPBS, 100 µL of a substrate solution [0.1 M citrate buffer (pH 4.0) containing 0.003 % H2O2 and 0.3 mg/mL of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (Wako Pure Chemical Ind., Ltd.)] was added and incubated for 15 min. The absorbance at 405 nm was measured using a microplate reader (Multiskan FC microplate photometer; Thermo Scientific). All reactions were performed at 37 °C.
Indirect competitive ELISA using anti-notoginseng-R1 monoclonal antibody An indirect competitive ELISA was performed as described above with slight modifications. After blocking the plate with SPBS and washing three times, 50 µL of NG-R1 or the ginseng samples at varying concentrations in 20 % MeOH were incubated for 1 hr
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Original Papers
with 50 µL of the mAb solution (7.4 µg/mL). The plate was washed three times with TPBS and combined with 100 µL of a 1 : 1000 dilution of peroxidase-anti-mouse IgG for 1 hr. Subsequently, 100 µL of the substrate solution was added to each well and incubated for 15 min. The absorbance was read at 405 nm. The CR of the NG‑R1 and related compounds was calculated using Weiler and Zenkʼs equation [21]: % Cross reactivity (CR) = [(concentration of NG‑R1 yielding A/ A0 = 50 %)/ (concentration of the related compound yielding A/A0 = 50 %)] × 100 where A is the absorbance in the presence of the test compound, and A0 is the absorbance in the absence of the test compound (20% MeOH solution).
Recovery of notoginseng-R1 from ginseng samples A Sanqi ginseng sample (36 mg) was extracted five times with 18 mL MeOH at a time using sonication. All extracts were combined and divided into 18 samples. Each ginseng extract sample (1 mL) was spiked with different amounts of a standard solution of NG‑R1: 25, 50, 75, 100, and 200 µg (n = 3). Each extract was evaporated to dryness and then dissolved in 1 mL MeOH. All samples were analyzed by the described ELISA method. For unspiked samples, a concentration of 211 µg/mL NG‑R1 was determined. The recovery (%) for spiked samples was calculated on the basis of the amount of measured and spiked NG‑R1: Recoveryð%Þ ¼
Measured amount of NR R1 211 100 Spiked amount of NG R1
Acknowledgments !
This study was supported, in part, by a Grant-in-Aid from JSPSʼs Asian CORE Program, the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the research fund of the Kyushu University Foundation and the Royal Golden Jubilee Ph. D Program, and the Thailand Research Fund (PHD/0311/2550).
Conflict of Interest !
The authors have declared no competing interests (financial or otherwise) related to this manuscript.
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