Chinese Journal of Natural Medicines 2014, 12(10): 07940799
Chinese Journal of Natural Medicines
A monoclonal antibody-based competitive ELISA for the determination of ruscogenin in Chinese traditional medicines and biological samples XU Yu1, LIU Ji-Hua1*, WANG Jing1, ZHANG Jian1, YU Bo-Yang2* 1
Department of Complex Prescription of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 211198, China;
2
Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Nanjing 210009, China Available online Octeber 2014 [ABSTRACT] A competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine ruscogenin (RUS) by using the monoclonal antibody (McAb). The monoclonal antibody against RUS, secreted from the established hybridoma cell lines, was identified as being of the IgG1 isotype. The McAb exhibited high specificity to RUS, showing a very slight cross reactivity with diosgenin (15.7%), and no cross-reactivity to sarsasapogenin, diammonium glycyrrhizinate, oleanolic acid and notoginsenoside R1. The established ELISA, at an IC50 value of 157.55 ng.mL−1 and a detection limit (IC20) of 20.57 ng.mL−1, was compared with HPLC analyses, and a good correlation between ELISA and HPLC-ELSD analyses of RUS in the extract of Radix Ophiopogonis was obtained. The experimental data indicated that the ELISA method exhibits more advantages over HPLC-ELSD, such as low detection limit, high specificity, low background, and no requirement for sample pre-treatment, and is more suitable for the determination of natural components in Chinese traditional medicines and in biological samples for pharmacokinetic studies.
[KEY WORDS] ELISA; Monoclonal antibody; Ruscogenin; Radix Ophiopogonis; Pharmacokinetic
[CLC Number] R917
[Document code] A
[Article ID] 2095-6975(2014)10-0794-06
Introduction Ruscogenin (RUS) is a steroidal sapogenin from the bulb stems of the Chinese edible plant Allium cepa L. var. agrogatum Don (Alliaceae) [1] and the traditional Chinese herb Radix Ophiopogonis (Ophiopogon japonicus (L. f.) Ker Gawl. (Asparagaceae), and has been clinically used for a long time to treat acute and chronic inflammatory and cardiovascular diseases. RUS has been found to exert significant anti-inflammatory activities in many aspects such as anti-elastase activity,
[Received on] 23-Aug.-2013 [Research funding] This project was supported by the Key Project of Chinese Ministry of Education (No. 108071), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and by the National Natural Science Foundation of China (No. 81173471). [*Corresponding author] LIU Ji-Hua: Prof., Tel/fax: 86-25-86185158, E-mail: [email protected]; YU Bo-Yang: Prof., boyangyu59@163. com These authors have no any conflict of interest to declare. Published by Elsevier B.V. All rights reserved
inhibiting leukocyte adhesion and migration, improving liver injury etc and anti-thrombotic activity [2-5]. As a potential therapeutic agent for acute lung injury or sepsis, RUS significantly attenuates LPS-induced acute lung injury by inhibiting expressions of TF and iNOS and NF-κB p65 activation [6]. Several analytical methods for the determination of RUS were developed, such as a colorimetric method, and HPLC or ultra performance liquid chromatography (UPLC) [7-8]. However, RUS is usually difficult to detect by HPLC–UV since it lacks a strong UV chromophore [8] and these assays are always characterized by low sensitivity or tedious pre-treatment before analysis, and are not suitable for the trace analysis of RUS. On the other hand, immunoassay has been proved to be a powerful tool for the trace analysis of natural products in complex matrices due to its outstanding sensitivity, remarkable specificity, high efficiency and can be used for the trace determination in pharmacokinetic studies [9]. In a previous study from this laboratory, an enzyme-linked immunosorbent assay (ELISA) based on the anti-ruscogenin polyclonal antibody has been established in our laboratory [10-11]. Monoclonal antibodies are often con-
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sidered superior to polyclonal antibodies because of their specificity to a single epitope, their homogeneous structure, and their ability to be mass-produced [12]. In this report, the monoclonal antibody against RUS is prepared and demonstrated to be a very useful tool to for the quality control or quantitative determination of RUS in crude Chinese medicine and rat serum.
Materials and Methods Reagents and immunochemicals RUS and the succinylated RUS-carrier protein conjugates (RUS-HS-BSA=OVA) were prepared in this laboratory [10-11]. Freund's complete or incomplete adjuvants was purchased from Sigma (St., MO, Louis, USA). Dulbecco’s Modified Eagle Medium (DMEM) and RPMI Medium 1640 were purchased from Gibco (Grand Island, NY, USA). Fetal Bovine Serum (FBS) was obtained from Hyclone (Logan, UT, USA). 3, 3', 5, 5'-Tetramethylbenzidine (TMB) was obtained from Amresco Chemical Co (Solon, OH, USA). Horseradish peroxidase labeled goat anti-mouse IgG and FITC-conjugated goat anti-mouse IgG were provided by Wuhan Boster Biological Technology Co., Ltd. (Wuhan, China). All other chemicals were standard commercial products of analytical grade. Materials and instruments. The Mouse Monoclonal Antibody Isotyping Kit was purchased from Sigma (St. Louis, MO, USA). The polystyrene 96-well plate were obtained from Costar Inc. (Cambridge, MA, USA). A microplate reader (Tecan, Austria) was used to measure immunoassay absorbance at 450 nm. Animals Five male Sprague-Dawley rats (250−280 g) were obtained from the Qinglong Mountain Animal Breeding Ground (Nanjing, China). Six-week-old Balb/c female mice were obtained from the Comparative Medicine Center of Yangzhou University (Yangzhou, China). Protocols for the animal studies were approved by the Regulations of Experimental Animal Administration, State Committee of Science and Technology of the People’s Republic of China. Immunization and cell fusion Balb/c mice (Six-week-old, female) were subcutaneously immunized with 50 μg of RUS-HS-BSA in physiological saline emulsified with an equal volume of Freund's complete adjuvant. two booster injections at the same dose of RUS-HS-BSA in Freund's incomplete adjuvant were used to immunize the mice at 2-week intervals. Four days prior to the day of fusion, the mouse with the highest antibody titer of serum was selected to give an intraperitoneal injection only with RUS-HS-BSA in PBS [13]. The hybridoma cells were acquired by fusion of the spleen cells from the immunized mouse and the mouse myeloma cells by PEG2000 as the regular process described by Zeng et al [14]. Those positive wells that exhibited positive competition with RUS were submitted to further subcloning by the limited dilution method [15]. The cloned
hybridoma cells were then cultured in DMEM containing 10% FBS. Production, purification and isotyping of monoclonal antibodies A large amount of monoclonal antibody was produced in vitro by culturing and expanding hybridoma cells, collecting the cellar supernatants. The expanded hybridoma cells were injected into the Balb/c mice abdomen for ascites. The McAb from the cellular supernatants and ascites were purified by the caprylic/ammonium sulfate precipitation method and stored at −20 °C for use. McAb isotyping was performed by using the Mouse Monoclonal Antibody Isotyping Kit and the operational procedure based on the manufacturer’s instructions. Indirect competitive enzyme-linked immunosorbent assay An indirect competitive ELISA (ic-ELISA) was developed to measure RUS binding and cross-reactivity (CR) to related compounds. The ELISA was carried out as follows: The wells of a 96-well immunoplate were coated overnight with a solution (100 µL/well) containing RUS-HS-OVA (10 µg.mL−1) in 0.05 mol.L−1 carbonate buffer (pH 9.6). The plate was washed three times with PBS-T (0.01 mol.L−1 phosphate-buffered saline, containing 0.05% Tween-20, pH 7.4) and treated with 150 µL of PBS-T (150 µL) containing 0.5% gelatin for 1 h at 37 °C to reduce non-specific adsorption. Fifty microliters of the McAb was added to each well followed by the addition of RUS or related competitor and incubated for 2 h, followed by three washes. The second antibody, goat anti-mouse IgG-HRP with 1 : 10 000 dilution in assay buffer was added to each well and incubated for 1 h at 37 °C. Subsequently, the plate was washed three times with PBS-T, TMB peroxidase substrate solution (100 µL) was added to each well, and after incubating for 20 min in the dark at room temperature, the reaction was immediately terminated by the addition of 2 mol·L−1 sulfuric acid (50 µL/well). The activity of enzyme bound to the plate was measured by a microplate reader at 450 nm. Cross-reactivity studies Cross-reactivity was extremely important to evaluate the specificity of anti-RUS McAb. The CR of the antibody with RUS, several RUS analogues including diosgenin, sarsasapogenin and diammonium glycyrrhizinate, notoginsenoside R1, and oleanolic acid (Figs. 1a–f) were determined by ic-ELISA. The specificity (CR%) of the ELISA procedure was calculated in accordance with the following formula: CR (%) = (IC50 RUS/IC50 competitor) ×100 [16]. IC50 is the RUS concentration reducing the ELISA maximum response to 50%. Here, the CR (%) of RUS was defined as 100%. Assay accuracy To affirm the accuracy and reliability of the ic-ELISA for the determination of RUS in Radix Ophiopogonis samples and in negative control plasma, Radix Ophiopogonis (1.5 g) from Sichuan was used in quadruplicate for recovery studies. After spiking pure RUS at concentration of 0, 0.675,
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1.125 and 1.575 mg·g−1 into the samples of Radix Ophiopogonis powder, the sample extracts were extracted with acid hydrolysis as described below and the RUS content in each sample was determined by the ic-ELISA. The recov-
ery of biological samples for RUS in rat plasma were prepared by adding standard RUS at concentrations of 10, 100 and 500 ng·mL −1 (n = 3) to negative control plasma using vortexing.
Fig. 1 Structures of (a) ruscogenin, (b) diosgenin, (c) sarsasapogenin, (d) oleanolic acid, (e) diammonium glycyrrhizinat, and (f) notoginsenoside R1
LC–ELSD analysis of RUS Based on a slight modification of the procedure used by the method of Tang [7], the LC quantification of all of the compounds was performed on a Shimadzu LC-10A system consisting of binary pump, thermostated column compartment and an Alltech 2000ES Evaporative Light Scattering Detector (ELSD, Alltech Co., Ltd.). A Hedera C18 column (250 mm × 4.6 mm, 5 μm, Hanbon Sci. & Tech.) was used for all chromatographic separations. Detection parameters were as follows: flow rate: 1.0 mL·min−1; methanol/water eluent (82 : 12). Sample preparation Dried samples (3 g) of Radix Ophiopogonis from Sichuan, Tianjin, Zhejiang, Hebei and Anhui Provinces in China were powdered and refluxed with methanol (80 mL) at 80 °C for 4 h. After filtration through a 0.45 µm filtration membrane, the extract was evaporated. Saponins were extracted from the above extract in water liquid–liquid extraction. For this, water saturated n-butanol (45 mL) was added to the separatory funnel and mixed. The upper organic layer (45 mL) was collected and evaporated to dryness. For the acquisition, of RUS, the extracts were treated with 7% sulfuric acid and refluxed at 100 °C for 5 h as previously reported [7]. After neutralization with 8% sodium hydroxide, the solution was extracted three times with chloroform, evaporated and then dissolved with methanol (1 mL). Pharmacokinetics Study Six male Wistar rats were administered at a dose of 25
mg.kg−1 RUS intragastrically. Orbital blood samples were collected in heparinized tubes at 0.5, 1, 2, 4, 6, 8, 10, 12, 24 and 48 h after administration. The plasma from each sample was immediately removed by centrifugation at 4 C for 10 min (3 000 r·min−1) and stored at −20 °C for future use. The concentrations of RUS in the rat plasma samples were determined with the ic- ELISA as described above.
Results The characteristics of McAb As shown in Figs. 2-1 and 2-2, the ELISA titers of the
Fig. 2-1 The dilution of anti-RUS McAb from cell culture supernatant, directly, as detected by indirect ELSA. The result shows that the titer of the McAb is 800
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ng·mL−1 to 5 μg·mL−1. Plotting logit (B/Bo) versus log standard RUS concentration yielded a linear response (R2 = 0.992 6; shown in Fig. 3).
Fig. 2-2 The dilution of anti-RUS McAb from purified ascites, indicating that the titer of McAb is 32000
McAb from cellar supernatants and purified ascites were 800 and 32 000, respectively. The McAb secreted by the hybridoma belongs to the IgG1 class. As shown in Table 1, the reactivity of McAb towards RUS was taken as 100% and cross-reacted with diosgenin (15.7%), sarsasapogenin (< 0.01%). The differences in C-1, C-5, and C-6 reduced the affinities of the McAb to these components. In addition, the antibody showed little cross-reactivity with diammonium glycyrrhizinate, oleanolic acid and notoginsenoside R1, all of which lack spiroketal structures. The reactivity towards RUS was referred to as 100%, suggesting that the McAb could be utilized for the determination of RUS concentration in Chinese traditional medicines and biological samples. Table 1 pounds
Fig. 3 Standard curves for the determination of ruscogenin in 10% methanol by ELISA with McAb. The insert shows the linearized calibration curve by the same experimental data. Various concentrations of RUS were incubated with McAb in a 96-well immunoplate with RUS-HS-OVA (10 μg·mL−1). B and Bo are the percentages of binding in the presence and absence of RUS, respectively. The logit-log plot is obtained from ln[(B/Bo)/(1−B/Bo)] Table 2 Recovery of RUS determined by ELISA in spiked samples (n = 3) Spiked level (mg·g−1)
Ruscogenin Diosgenin
−1
IC50 (ng·mL ) Cross reactivities/% 144.92 918.97
100
> 10 000
< 0.01
Diammoniumglycyrrhizinate
> 10 000
< 0.01
Notoginsenoside R1
> 10 000
< 0.01
Oleanolic acid
> 10 000
< 0.01
Recovery (%)c
1.13 ± 0.06
5.31
0.675
1.92 ± 0.13
6.77
117.04
1.125
2.16 ± 0.10
4.63
91.56
1.575
2.68 ± 0.19
7.09
98.41
a
RUS in spiked samples was extracted with sample preparation. Data were mean ± SD from triplicate samples for each level; b Coeffcient of variation = SD/ mean × 100%; c The percentage of recovery was calculated as follows: Recovery (%) = (measured amount － 1.13)/spiked amount × 100
15.77
Sarsasapogenin
CV (%)b
0
Cross reactivities of McAb against various com-
Compounds
Measured amount (mg·g−1)a
Evaluation of the ELISA In the ELISA, the preliminary findings revealed that 10 μg·mL−1 of RUS-HS-OVA was the minimum concentration required to produce an appropriate color after application of a chromogenic substrate. Under the above conditions of 10 μg·mL−1 of RUS-HS-OVA as the coating solution, a 1 000 times diluted solution of the purified McAb (1.96 μg·mL−1), which produced an OD of about 1.0, was selected for the ELISA to detect RUS. After the assay procedure was optimized in the ELISA, binding of McAb to a solid-phase RUS-HS-OVA conjugate was inhibited in the range of 10
Recoveries of RUS extracted from Radix Ophiopogonis spiked at three dose levels were shown in Table 2, and good RUS recoveries were shown ranging from 91.56% to 117.04% with 4.63%−7.09% of CV. As shown in Table 3, the accuracy of the intra- and inter-assays in negative control plasma were in a range of 97.05%−111.40% (n = 6) and 94.95%−101.20%. The coefficients of variance for intra- and inter-assays were 4.88% to 11.79% and 2.88% to 4.99%, respectively. These data indicated that the recoveries of RUS spiked in Radix Ophiopogonis samples and in negative control plasma were within an acceptable range with deviations from a mean also within an acceptable range. It can be concluded that the ELISA using the McAb can be used for the reliable determination of RUS.
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Correlation of RUS content in plant samples determined by ELISA and HPLC Compared with the indirect competitive ELISA method, a HPLC-ELSD method was performed by determining the concentrations of RUS in the extract of Radix Ophiopogonis as indicated in Table 4. The correlation coefficient was Table 3 Precision and accuracy of ELISA for RUS in rat plasma Add (ng.mL−1)
Estimated (ng.mL−1)a
Recovery/ %b
CV/ %c
500
515.86 ± 60.86
103.17
11.79
100
97.05 ± 4.74
97.05
4.88
Intra-assay
10
11.14 ± 1.09
111.40
9.78
500
506.00 ± 25.27
101.20
4.99
100
94.95 ± 2.74
94.95
2.88
10
9.90 ± 0.45
99.00
4.55
Inter-assay a
Values represent the mean ± SD of 6 experiments; b Recovery = measured [RUS]/spiked[RUS] × 100%; c Coeffcient of variation
derived from fitting a straight line analyzed by ELISA and HPLC methods as shown in Fig. 4 [17-18]. There was a good correlation between the values determined by the two methods and the correlation coefficient was 0.999 4. Application to RUS pharmacokinetics evaluation in rat plasma The presently established ELISA was applied to the pharmacokinetic evaluation of RUS in rats for further validation of its reliability. The negative control serum used to prepare PPT standards for calibration curves did not alter the working range of this method (data not shown). The mean plasma concentration-time curve was illustrated in Fig. 5. The pharmacokinetic parameters were calculated using DAS 2.0 and the data were summarized in Table 5. RUS was characterized by a relatively long terminal elimination phase half-life (> 6 h). These results indicated that the established ELISA method was sufficiently sensitive and reliable for the study of RUS pharmacokinetics.
Table 4 Total concentration of RUS in Radix Ophiopogonis samples determined by HPLC and ELISA Total concentration of ruscogenin from different locations (mg.g·1) Method
Sichuan
Tianjian
Zhejiang
Hebei
Anhui
ELISA
2.55 ± 0.03
1.9 ± 0.03
0.83 ± 0.05
1.51 ± 0.08
1.36 ± 0.04
HPLC-ELSD
3.28 ± 0.01
2.4 ± 0.02
1.04 ± 0.02
1.89 ± 0.01
1.69 ± 0.01
Table 5 Pharmacokinetic parameters of RUS in 5 rats after an oral dose of 25 mg.kg−1 Parameter
Value
−1
−1
52 099.59 ± 27 783.53
−1
−1
54 131.17 ± 28 829.49
AUC(0-t)(μg·L ・h ) AUC(0-∞)(μg·L ・h ) MRT(0-t)/h
13.88 ± 3.05
MRT(0-∞)/h
16.48 ± 6.44
t1/2/h
7.99 ± 3.31
tmax/h
9.60 ± 2.19
CLz/F(L·h−1·kg−1) −1
cmax(μg·L )
Fig. 4 Correlation between RUS values determined by ELISA and HPLC in spiked samples
0.77 ± 0.80 3 327.15 ± 1 845.29
Discussion In general, the spiroketal structures are regarded as antigenic determinants that play an important role in the specificity of McAb and the McAb can be used for the determination of steroidal aglycones. Meanwhile, McAb can be produced in the same quality at any time, which is important for the stability of an assay system, whereas a polyclonal antibody cannot be expected to have such a character. As the major bioactive steroidal aglycone found in Radix Ophiopogonis, the concentration of RUS can be detected by the
Fig. 5 Mean plasma concentration-time profiles of RUS after a single oral administration to rats (n = 5)
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ELISA based on the McAb. Quantification of the active components in Chinese traditional medicine is very important for their quality control. In the past years, HPLC methods have been applied frequently in such a field. However, because of the complexity and the limitation that an active component, such as RUS, lacks a strong UV, these methods have shown some disadvantages for the analysis of crude drugs, such as low specificity and requirements of sample pre-treatment. The ELISA for the determination of RUS in Radix Ophiopogonis from various areas provides a simple, fast, and useful tool for the quality control. The good correlation coefficient of 0.999 4 also supports the use of ELSIA for the determination of RUS. Since the practical measuring range of the HPLC- ELSD method performed in this paper was 50 to 600 μg.mL−1, it is evident that the ELISA with a range of 10 ng·mL−1 to 5 μg·mL−1, is approximately 100 times more sensitive. This indicated that the ELISA can be applied for the trace analysis of RUS in crude plants and biological samples for pharmacokinetics, without the need for tedious pre-treatment before analysis. Furthermore, the satisfactory recovery rates obtained by ELISA in rat plasma established that the ELISA method was also suitable for the analysis of biological samples. A preliminary pharmacokinetic study of RUS in rats validated such a use. After oral administration of RUS, the compound was detectable in plasma at fixed points in 48 h, which suggested that it was very easy for RUS to be absorbed from the intestine and may not be transformed or decomposed in vivo. In conclusion, the ELISA procedure for RUS reported here is sensitive, specific, reproducible, simple, and adaptable for the analysis of a large number of Radix Ophiopogonis and biological samples in pharmacokinetic studies.
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Cite this article as: XU Yu, LIU Ji-Hua, WANG Jing, ZHANG Jian, YU Bo-Yang. A monoclonal antibody-based competitive ELISA for the determination of ruscogenin in Chinese traditional medicines and biological samples [J]. Chinese Journal of Natural Medicines, 2014, 12 (10): 794-799.
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Chinese Journal of Natural Medicines
A monoclonal antibody-based competitive ELISA for the determination of ruscogenin in Chinese traditional medicines and biological samples XU Yu1, LIU Ji-Hua1*, WANG Jing1, ZHANG Jian1, YU Bo-Yang2* 1
Department of Complex Prescription of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 211198, China;
2
Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Nanjing 210009, China Available online Octeber 2014 [ABSTRACT] A competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine ruscogenin (RUS) by using the monoclonal antibody (McAb). The monoclonal antibody against RUS, secreted from the established hybridoma cell lines, was identified as being of the IgG1 isotype. The McAb exhibited high specificity to RUS, showing a very slight cross reactivity with diosgenin (15.7%), and no cross-reactivity to sarsasapogenin, diammonium glycyrrhizinate, oleanolic acid and notoginsenoside R1. The established ELISA, at an IC50 value of 157.55 ng.mL−1 and a detection limit (IC20) of 20.57 ng.mL−1, was compared with HPLC analyses, and a good correlation between ELISA and HPLC-ELSD analyses of RUS in the extract of Radix Ophiopogonis was obtained. The experimental data indicated that the ELISA method exhibits more advantages over HPLC-ELSD, such as low detection limit, high specificity, low background, and no requirement for sample pre-treatment, and is more suitable for the determination of natural components in Chinese traditional medicines and in biological samples for pharmacokinetic studies.
[KEY WORDS] ELISA; Monoclonal antibody; Ruscogenin; Radix Ophiopogonis; Pharmacokinetic
[CLC Number] R917
[Document code] A
[Article ID] 2095-6975(2014)10-0794-06
Introduction Ruscogenin (RUS) is a steroidal sapogenin from the bulb stems of the Chinese edible plant Allium cepa L. var. agrogatum Don (Alliaceae) [1] and the traditional Chinese herb Radix Ophiopogonis (Ophiopogon japonicus (L. f.) Ker Gawl. (Asparagaceae), and has been clinically used for a long time to treat acute and chronic inflammatory and cardiovascular diseases. RUS has been found to exert significant anti-inflammatory activities in many aspects such as anti-elastase activity,
[Received on] 23-Aug.-2013 [Research funding] This project was supported by the Key Project of Chinese Ministry of Education (No. 108071), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and by the National Natural Science Foundation of China (No. 81173471). [*Corresponding author] LIU Ji-Hua: Prof., Tel/fax: 86-25-86185158, E-mail: [email protected]; YU Bo-Yang: Prof., boyangyu59@163. com These authors have no any conflict of interest to declare. Published by Elsevier B.V. All rights reserved
inhibiting leukocyte adhesion and migration, improving liver injury etc and anti-thrombotic activity [2-5]. As a potential therapeutic agent for acute lung injury or sepsis, RUS significantly attenuates LPS-induced acute lung injury by inhibiting expressions of TF and iNOS and NF-κB p65 activation [6]. Several analytical methods for the determination of RUS were developed, such as a colorimetric method, and HPLC or ultra performance liquid chromatography (UPLC) [7-8]. However, RUS is usually difficult to detect by HPLC–UV since it lacks a strong UV chromophore [8] and these assays are always characterized by low sensitivity or tedious pre-treatment before analysis, and are not suitable for the trace analysis of RUS. On the other hand, immunoassay has been proved to be a powerful tool for the trace analysis of natural products in complex matrices due to its outstanding sensitivity, remarkable specificity, high efficiency and can be used for the trace determination in pharmacokinetic studies [9]. In a previous study from this laboratory, an enzyme-linked immunosorbent assay (ELISA) based on the anti-ruscogenin polyclonal antibody has been established in our laboratory [10-11]. Monoclonal antibodies are often con-
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XU Yu, et al. / Chin J Nat Med, 2014, 12(10): 794799
sidered superior to polyclonal antibodies because of their specificity to a single epitope, their homogeneous structure, and their ability to be mass-produced [12]. In this report, the monoclonal antibody against RUS is prepared and demonstrated to be a very useful tool to for the quality control or quantitative determination of RUS in crude Chinese medicine and rat serum.
Materials and Methods Reagents and immunochemicals RUS and the succinylated RUS-carrier protein conjugates (RUS-HS-BSA=OVA) were prepared in this laboratory [10-11]. Freund's complete or incomplete adjuvants was purchased from Sigma (St., MO, Louis, USA). Dulbecco’s Modified Eagle Medium (DMEM) and RPMI Medium 1640 were purchased from Gibco (Grand Island, NY, USA). Fetal Bovine Serum (FBS) was obtained from Hyclone (Logan, UT, USA). 3, 3', 5, 5'-Tetramethylbenzidine (TMB) was obtained from Amresco Chemical Co (Solon, OH, USA). Horseradish peroxidase labeled goat anti-mouse IgG and FITC-conjugated goat anti-mouse IgG were provided by Wuhan Boster Biological Technology Co., Ltd. (Wuhan, China). All other chemicals were standard commercial products of analytical grade. Materials and instruments. The Mouse Monoclonal Antibody Isotyping Kit was purchased from Sigma (St. Louis, MO, USA). The polystyrene 96-well plate were obtained from Costar Inc. (Cambridge, MA, USA). A microplate reader (Tecan, Austria) was used to measure immunoassay absorbance at 450 nm. Animals Five male Sprague-Dawley rats (250−280 g) were obtained from the Qinglong Mountain Animal Breeding Ground (Nanjing, China). Six-week-old Balb/c female mice were obtained from the Comparative Medicine Center of Yangzhou University (Yangzhou, China). Protocols for the animal studies were approved by the Regulations of Experimental Animal Administration, State Committee of Science and Technology of the People’s Republic of China. Immunization and cell fusion Balb/c mice (Six-week-old, female) were subcutaneously immunized with 50 μg of RUS-HS-BSA in physiological saline emulsified with an equal volume of Freund's complete adjuvant. two booster injections at the same dose of RUS-HS-BSA in Freund's incomplete adjuvant were used to immunize the mice at 2-week intervals. Four days prior to the day of fusion, the mouse with the highest antibody titer of serum was selected to give an intraperitoneal injection only with RUS-HS-BSA in PBS [13]. The hybridoma cells were acquired by fusion of the spleen cells from the immunized mouse and the mouse myeloma cells by PEG2000 as the regular process described by Zeng et al [14]. Those positive wells that exhibited positive competition with RUS were submitted to further subcloning by the limited dilution method [15]. The cloned
hybridoma cells were then cultured in DMEM containing 10% FBS. Production, purification and isotyping of monoclonal antibodies A large amount of monoclonal antibody was produced in vitro by culturing and expanding hybridoma cells, collecting the cellar supernatants. The expanded hybridoma cells were injected into the Balb/c mice abdomen for ascites. The McAb from the cellular supernatants and ascites were purified by the caprylic/ammonium sulfate precipitation method and stored at −20 °C for use. McAb isotyping was performed by using the Mouse Monoclonal Antibody Isotyping Kit and the operational procedure based on the manufacturer’s instructions. Indirect competitive enzyme-linked immunosorbent assay An indirect competitive ELISA (ic-ELISA) was developed to measure RUS binding and cross-reactivity (CR) to related compounds. The ELISA was carried out as follows: The wells of a 96-well immunoplate were coated overnight with a solution (100 µL/well) containing RUS-HS-OVA (10 µg.mL−1) in 0.05 mol.L−1 carbonate buffer (pH 9.6). The plate was washed three times with PBS-T (0.01 mol.L−1 phosphate-buffered saline, containing 0.05% Tween-20, pH 7.4) and treated with 150 µL of PBS-T (150 µL) containing 0.5% gelatin for 1 h at 37 °C to reduce non-specific adsorption. Fifty microliters of the McAb was added to each well followed by the addition of RUS or related competitor and incubated for 2 h, followed by three washes. The second antibody, goat anti-mouse IgG-HRP with 1 : 10 000 dilution in assay buffer was added to each well and incubated for 1 h at 37 °C. Subsequently, the plate was washed three times with PBS-T, TMB peroxidase substrate solution (100 µL) was added to each well, and after incubating for 20 min in the dark at room temperature, the reaction was immediately terminated by the addition of 2 mol·L−1 sulfuric acid (50 µL/well). The activity of enzyme bound to the plate was measured by a microplate reader at 450 nm. Cross-reactivity studies Cross-reactivity was extremely important to evaluate the specificity of anti-RUS McAb. The CR of the antibody with RUS, several RUS analogues including diosgenin, sarsasapogenin and diammonium glycyrrhizinate, notoginsenoside R1, and oleanolic acid (Figs. 1a–f) were determined by ic-ELISA. The specificity (CR%) of the ELISA procedure was calculated in accordance with the following formula: CR (%) = (IC50 RUS/IC50 competitor) ×100 [16]. IC50 is the RUS concentration reducing the ELISA maximum response to 50%. Here, the CR (%) of RUS was defined as 100%. Assay accuracy To affirm the accuracy and reliability of the ic-ELISA for the determination of RUS in Radix Ophiopogonis samples and in negative control plasma, Radix Ophiopogonis (1.5 g) from Sichuan was used in quadruplicate for recovery studies. After spiking pure RUS at concentration of 0, 0.675,
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1.125 and 1.575 mg·g−1 into the samples of Radix Ophiopogonis powder, the sample extracts were extracted with acid hydrolysis as described below and the RUS content in each sample was determined by the ic-ELISA. The recov-
ery of biological samples for RUS in rat plasma were prepared by adding standard RUS at concentrations of 10, 100 and 500 ng·mL −1 (n = 3) to negative control plasma using vortexing.
Fig. 1 Structures of (a) ruscogenin, (b) diosgenin, (c) sarsasapogenin, (d) oleanolic acid, (e) diammonium glycyrrhizinat, and (f) notoginsenoside R1
LC–ELSD analysis of RUS Based on a slight modification of the procedure used by the method of Tang [7], the LC quantification of all of the compounds was performed on a Shimadzu LC-10A system consisting of binary pump, thermostated column compartment and an Alltech 2000ES Evaporative Light Scattering Detector (ELSD, Alltech Co., Ltd.). A Hedera C18 column (250 mm × 4.6 mm, 5 μm, Hanbon Sci. & Tech.) was used for all chromatographic separations. Detection parameters were as follows: flow rate: 1.0 mL·min−1; methanol/water eluent (82 : 12). Sample preparation Dried samples (3 g) of Radix Ophiopogonis from Sichuan, Tianjin, Zhejiang, Hebei and Anhui Provinces in China were powdered and refluxed with methanol (80 mL) at 80 °C for 4 h. After filtration through a 0.45 µm filtration membrane, the extract was evaporated. Saponins were extracted from the above extract in water liquid–liquid extraction. For this, water saturated n-butanol (45 mL) was added to the separatory funnel and mixed. The upper organic layer (45 mL) was collected and evaporated to dryness. For the acquisition, of RUS, the extracts were treated with 7% sulfuric acid and refluxed at 100 °C for 5 h as previously reported [7]. After neutralization with 8% sodium hydroxide, the solution was extracted three times with chloroform, evaporated and then dissolved with methanol (1 mL). Pharmacokinetics Study Six male Wistar rats were administered at a dose of 25
mg.kg−1 RUS intragastrically. Orbital blood samples were collected in heparinized tubes at 0.5, 1, 2, 4, 6, 8, 10, 12, 24 and 48 h after administration. The plasma from each sample was immediately removed by centrifugation at 4 C for 10 min (3 000 r·min−1) and stored at −20 °C for future use. The concentrations of RUS in the rat plasma samples were determined with the ic- ELISA as described above.
Results The characteristics of McAb As shown in Figs. 2-1 and 2-2, the ELISA titers of the
Fig. 2-1 The dilution of anti-RUS McAb from cell culture supernatant, directly, as detected by indirect ELSA. The result shows that the titer of the McAb is 800
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ng·mL−1 to 5 μg·mL−1. Plotting logit (B/Bo) versus log standard RUS concentration yielded a linear response (R2 = 0.992 6; shown in Fig. 3).
Fig. 2-2 The dilution of anti-RUS McAb from purified ascites, indicating that the titer of McAb is 32000
McAb from cellar supernatants and purified ascites were 800 and 32 000, respectively. The McAb secreted by the hybridoma belongs to the IgG1 class. As shown in Table 1, the reactivity of McAb towards RUS was taken as 100% and cross-reacted with diosgenin (15.7%), sarsasapogenin (< 0.01%). The differences in C-1, C-5, and C-6 reduced the affinities of the McAb to these components. In addition, the antibody showed little cross-reactivity with diammonium glycyrrhizinate, oleanolic acid and notoginsenoside R1, all of which lack spiroketal structures. The reactivity towards RUS was referred to as 100%, suggesting that the McAb could be utilized for the determination of RUS concentration in Chinese traditional medicines and biological samples. Table 1 pounds
Fig. 3 Standard curves for the determination of ruscogenin in 10% methanol by ELISA with McAb. The insert shows the linearized calibration curve by the same experimental data. Various concentrations of RUS were incubated with McAb in a 96-well immunoplate with RUS-HS-OVA (10 μg·mL−1). B and Bo are the percentages of binding in the presence and absence of RUS, respectively. The logit-log plot is obtained from ln[(B/Bo)/(1−B/Bo)] Table 2 Recovery of RUS determined by ELISA in spiked samples (n = 3) Spiked level (mg·g−1)
Ruscogenin Diosgenin
−1
IC50 (ng·mL ) Cross reactivities/% 144.92 918.97
100
> 10 000
< 0.01
Diammoniumglycyrrhizinate
> 10 000
< 0.01
Notoginsenoside R1
> 10 000
< 0.01
Oleanolic acid
> 10 000
< 0.01
Recovery (%)c
1.13 ± 0.06
5.31
0.675
1.92 ± 0.13
6.77
117.04
1.125
2.16 ± 0.10
4.63
91.56
1.575
2.68 ± 0.19
7.09
98.41
a
RUS in spiked samples was extracted with sample preparation. Data were mean ± SD from triplicate samples for each level; b Coeffcient of variation = SD/ mean × 100%; c The percentage of recovery was calculated as follows: Recovery (%) = (measured amount － 1.13)/spiked amount × 100
15.77
Sarsasapogenin
CV (%)b
0
Cross reactivities of McAb against various com-
Compounds
Measured amount (mg·g−1)a
Evaluation of the ELISA In the ELISA, the preliminary findings revealed that 10 μg·mL−1 of RUS-HS-OVA was the minimum concentration required to produce an appropriate color after application of a chromogenic substrate. Under the above conditions of 10 μg·mL−1 of RUS-HS-OVA as the coating solution, a 1 000 times diluted solution of the purified McAb (1.96 μg·mL−1), which produced an OD of about 1.0, was selected for the ELISA to detect RUS. After the assay procedure was optimized in the ELISA, binding of McAb to a solid-phase RUS-HS-OVA conjugate was inhibited in the range of 10
Recoveries of RUS extracted from Radix Ophiopogonis spiked at three dose levels were shown in Table 2, and good RUS recoveries were shown ranging from 91.56% to 117.04% with 4.63%−7.09% of CV. As shown in Table 3, the accuracy of the intra- and inter-assays in negative control plasma were in a range of 97.05%−111.40% (n = 6) and 94.95%−101.20%. The coefficients of variance for intra- and inter-assays were 4.88% to 11.79% and 2.88% to 4.99%, respectively. These data indicated that the recoveries of RUS spiked in Radix Ophiopogonis samples and in negative control plasma were within an acceptable range with deviations from a mean also within an acceptable range. It can be concluded that the ELISA using the McAb can be used for the reliable determination of RUS.
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Correlation of RUS content in plant samples determined by ELISA and HPLC Compared with the indirect competitive ELISA method, a HPLC-ELSD method was performed by determining the concentrations of RUS in the extract of Radix Ophiopogonis as indicated in Table 4. The correlation coefficient was Table 3 Precision and accuracy of ELISA for RUS in rat plasma Add (ng.mL−1)
Estimated (ng.mL−1)a
Recovery/ %b
CV/ %c
500
515.86 ± 60.86
103.17
11.79
100
97.05 ± 4.74
97.05
4.88
Intra-assay
10
11.14 ± 1.09
111.40
9.78
500
506.00 ± 25.27
101.20
4.99
100
94.95 ± 2.74
94.95
2.88
10
9.90 ± 0.45
99.00
4.55
Inter-assay a
Values represent the mean ± SD of 6 experiments; b Recovery = measured [RUS]/spiked[RUS] × 100%; c Coeffcient of variation
derived from fitting a straight line analyzed by ELISA and HPLC methods as shown in Fig. 4 [17-18]. There was a good correlation between the values determined by the two methods and the correlation coefficient was 0.999 4. Application to RUS pharmacokinetics evaluation in rat plasma The presently established ELISA was applied to the pharmacokinetic evaluation of RUS in rats for further validation of its reliability. The negative control serum used to prepare PPT standards for calibration curves did not alter the working range of this method (data not shown). The mean plasma concentration-time curve was illustrated in Fig. 5. The pharmacokinetic parameters were calculated using DAS 2.0 and the data were summarized in Table 5. RUS was characterized by a relatively long terminal elimination phase half-life (> 6 h). These results indicated that the established ELISA method was sufficiently sensitive and reliable for the study of RUS pharmacokinetics.
Table 4 Total concentration of RUS in Radix Ophiopogonis samples determined by HPLC and ELISA Total concentration of ruscogenin from different locations (mg.g·1) Method
Sichuan
Tianjian
Zhejiang
Hebei
Anhui
ELISA
2.55 ± 0.03
1.9 ± 0.03
0.83 ± 0.05
1.51 ± 0.08
1.36 ± 0.04
HPLC-ELSD
3.28 ± 0.01
2.4 ± 0.02
1.04 ± 0.02
1.89 ± 0.01
1.69 ± 0.01
Table 5 Pharmacokinetic parameters of RUS in 5 rats after an oral dose of 25 mg.kg−1 Parameter
Value
−1
−1
52 099.59 ± 27 783.53
−1
−1
54 131.17 ± 28 829.49
AUC(0-t)(μg·L ・h ) AUC(0-∞)(μg·L ・h ) MRT(0-t)/h
13.88 ± 3.05
MRT(0-∞)/h
16.48 ± 6.44
t1/2/h
7.99 ± 3.31
tmax/h
9.60 ± 2.19
CLz/F(L·h−1·kg−1) −1
cmax(μg·L )
Fig. 4 Correlation between RUS values determined by ELISA and HPLC in spiked samples
0.77 ± 0.80 3 327.15 ± 1 845.29
Discussion In general, the spiroketal structures are regarded as antigenic determinants that play an important role in the specificity of McAb and the McAb can be used for the determination of steroidal aglycones. Meanwhile, McAb can be produced in the same quality at any time, which is important for the stability of an assay system, whereas a polyclonal antibody cannot be expected to have such a character. As the major bioactive steroidal aglycone found in Radix Ophiopogonis, the concentration of RUS can be detected by the
Fig. 5 Mean plasma concentration-time profiles of RUS after a single oral administration to rats (n = 5)
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ELISA based on the McAb. Quantification of the active components in Chinese traditional medicine is very important for their quality control. In the past years, HPLC methods have been applied frequently in such a field. However, because of the complexity and the limitation that an active component, such as RUS, lacks a strong UV, these methods have shown some disadvantages for the analysis of crude drugs, such as low specificity and requirements of sample pre-treatment. The ELISA for the determination of RUS in Radix Ophiopogonis from various areas provides a simple, fast, and useful tool for the quality control. The good correlation coefficient of 0.999 4 also supports the use of ELSIA for the determination of RUS. Since the practical measuring range of the HPLC- ELSD method performed in this paper was 50 to 600 μg.mL−1, it is evident that the ELISA with a range of 10 ng·mL−1 to 5 μg·mL−1, is approximately 100 times more sensitive. This indicated that the ELISA can be applied for the trace analysis of RUS in crude plants and biological samples for pharmacokinetics, without the need for tedious pre-treatment before analysis. Furthermore, the satisfactory recovery rates obtained by ELISA in rat plasma established that the ELISA method was also suitable for the analysis of biological samples. A preliminary pharmacokinetic study of RUS in rats validated such a use. After oral administration of RUS, the compound was detectable in plasma at fixed points in 48 h, which suggested that it was very easy for RUS to be absorbed from the intestine and may not be transformed or decomposed in vivo. In conclusion, the ELISA procedure for RUS reported here is sensitive, specific, reproducible, simple, and adaptable for the analysis of a large number of Radix Ophiopogonis and biological samples in pharmacokinetic studies.
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Cite this article as: XU Yu, LIU Ji-Hua, WANG Jing, ZHANG Jian, YU Bo-Yang. A monoclonal antibody-based competitive ELISA for the determination of ruscogenin in Chinese traditional medicines and biological samples [J]. Chinese Journal of Natural Medicines, 2014, 12 (10): 794-799.
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