J. Sep. Sci. 2015, 38, 2363–2370
Yue Zhang1 ∗ Huihua Qu2 ∗ Wenhao Zeng3 Yan Zhao4 Wenchao Shan3 Xueqian Wang4 Qingguo Wang4 Yan Zhao4 1 Department
of Biopharmaceutics, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China 2 Department of Molecular Biology, Centre of Scientific Experiment, Beijing University of Chinese Medicine, Beijing, China 3 Department of Chemistry of TCM, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China 4 Department of Basic TCM Clinical Science, School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing, China Received March 2, 2015 Revised April 8, 2015 Accepted April 15, 2015
Development of an enzyme-linked immunosorbent assay and immunoaffinity chromatography for glycyrrhizic acid using an anti-glycyrrhizic acid monoclonal antibody In this work, a new monoclonal antibody specific for glycyrrhizic acid was prepared and characterized. A hybridoma secreting an anti-glycyrrhizic acid monoclonal antibody was produced by fusing splenocytes from a mouse immunized against a glycyrrhizic acid– bovine serum albumin conjugate with the hypoxanthine–aminopterin–thymidine-sensitive mouse myeloma cell line (Sp2/0-Ag14). Subsequently, an indirect, competitive enzymelinked immunosorbent assay for glycyrrhizic acid was developed using the monoclonal antibody. In this assay, we detected an effective measuring range of 78.12–2500 ng/mL. Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10%. In addition, glycyrrhizic acid levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. We then successfully developed a reliable immunoaffinity chromatography to separate glycyrrhizic acid completely from its parent medicine. These methods will contribute to further research investigations to better understand the interactions of glycyrrhizic acid with other drugs in the complex system of traditional Chinese medicine. Keywords: Glycyrrhizic acid / Monoclonal antibodies / Indirect competitive enzyme-linked immunosorbent assay / Immunoaffinity chromatography DOI 10.1002/jssc.201500242
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Glycyrrhiza uralensis is a unique herb in traditional Chinese medicine (TCM). It is a component in most prescriptions and is considered to play a role in balancing the efficacy and safety of the formula . Glycyrrhizic acid (GA), which is the main active component of Glycyrrhiza uralensis, has been shown to have a variety of biological activities, such as anti-inflammatory [2, 3], anti-oxidation , anti-viral [5, 6] immunoregulation , and liver protection . Currently, GA and glycyrrhetate are used clinically to decrease liver damage  and reverse cisplatin resistance of hepatocellular Correspondence: Dr. Y. Zhao, Beijing University of Chinese Medicine, School of Basic Medical Sciences, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029,China E-mail: [email protected]
Fax: +86 1 6428 6821
Abbreviations: CBS, carbonate buffer solution; HRP, horseradish peroxidase; IAC, immunoaffinity chromatography; MAb, monoclonal antibody; MIP, molecularly imprinted polymer; GA, glycyrrhizic acid; OD, optical density; OVA, ovalbumin; PBS, phosphate-buffer saline; TCM, traditional Chinese medicine; TMB, tetramethyl benzidine C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
carcinoma cells through the inhibition of multi-drug resistance-associated proteins . Therefore, rapid and sensitive measurement of the content of GA in various samples is necessary. To this end, HPLC , GC–MS , LC–MS , and eastern blotting  methods have been used to quantify GA levels in medicine as well as biological samples. These methods are widely applied but the complicated pretreatment steps may be problematic for in vivo metabolism research. Recently, the monoclonal antibody (MAb)-based ELISA has become an emerging platform for the qualitative or quantitative analysis of food or natural products. This method is an accurate, sensitive, and effective qualitative method that does not include tedious pretreatment steps , thus it is essential for biological samples and future clinical applications. Though some MAbs against GA and MAb-based immunoassays have been reported [14, 16, 17], we provide an alternative ELISA with different sensitivity and specificity by a new MAb in this work. Up to now, the extraction and separation of GA from medicinal preparations are also important issues for further clinical research to study the mechanisms underlying the ∗ These
two authors contributed equally to this work. www.jss-journal.com
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effects of GA from other perspectives. In recent years, to examine the compatibility rule of complex prescriptions on the molecular level, much research has focused on the strategy of “target constituent knock-out”.  To separate the particular components in samples, many methods include TLC , preparative HPLC , HSCCC , and Fourier transform ion cyclotron resonance mass spectrometry  are reported. TLC could only be applied to ingredients that comprise a large percentage of the content due to complicated process. While the other methods rely on precise instruments and require significant investment to concentrate compounds in a large scale. Compared to conventional methods, one-step immunoaffinity chromatography (IAC) is considered to be more concise, cheaper, and time-saving strategy which is not dependent upon expensive instruments [21–23]. Furthermore, it is environmentally friendly since no organic reagents are used in the extraction process. Also, due to its sensitivity, the IAC column could capture the target compounds and concentrate them by repeated capture. IAC is therefore a promising new method for separation and purification of target compounds. Thus in this study, we first investigated the formation and characterization of anti-GC MAb, and then applied it in the development of an ELISA and an IAC method for detection and separation of GA from its structurally related compounds.
2 Materials and methods 2.1 Chemicals and reagents BSA, ovalbumin (OVA), and Freund’s complete and incomplete adjuvants were obtained from Sigma–Aldrich (St. Louis, MO, USA). GA and other Chinese herbal medicines were purchased from the National Institutes for Food and Drug Control (NIFDC, Beijing, China), and all had a purity of 98%. 3,3,5,5-Tetramethylbenzidine (TMB) was supplied by Sigma (St. Louis, MO, USA). Peroxidase-labeled anti-mouse IgG was provided by Organon Teknika Cappel Products (West Chester, PA, USA). Chinese medicines used in this paper were obtained from Beijing Tong Ren Tang Group (Beijing, China). All other analytical-grade chemical reagents were obtained from Sinopharm Chemical Reagents Beijing (Beijing, China). CNBr-activated Sepharose 4B were purchased from Pharmacia GE (Uppsala, Sweden).
J. Sep. Sci. 2015, 38, 2363–2370
from USTC Zonkia Scientific Instruments (Anhui, China). All 96-well immune plates were purchased from Corning Incorporated (Corning, NY, USA).
2.3 Synthesis of antigen conjugate Glycyrrhizic acid-BSA conjugates (GA-BSA) were synthesized using a periodate oxidation procedure based on a previously reported method with some modifications [23–25]. Briefly, 50 mg GA was dissolved in water to a concentration of 1 mg/mL. Then, 1 mL of a freshly prepared sodium periodate solution (0.1 M) was added dropwise into 50 mL of the GA solution. The mixture was stirred at room temperature for 1 h. Carbonate buffer (50 mM, pH 9.6, 2.0 mL) containing BSA (40 mg) was added to the above reaction mixture. The whole mixture was adjusted to pH 9 with 1 M Na2 CO3 solution and stirred at room temperature for an additional 6 h. The reaction mixture was dialyzed six times against phosphate-buffered saline (PBS) for three days. GA-OVA was synthesized in a similar manner.
2.4 Immunization and hybridization BALB/c female mice (eight weeks of age) were immunized with the GA-BSA conjugate, and MAb were generated as previously described  with some modifications. The first immunization (50 g conjugate per injection) consisted of a subcutaneous multipoint injection in the back using a 1:1 emulsion of the conjugate to Freund’s complete adjuvant. The subsequent immunizations (50 g conjugate per injection) were administered as a 1:1 emulsion in Freund’s incomplete adjuvant and performed at two week intervals. At the fourth and final immunization, a booster dose of 100 g of conjugate dissolved in PBS without adjuvant was administered intraperitoneally. Blood from the mice was obtained from the tail vein, and sera were tested for their titer by indirect ELISAs using GA-OVA as a solid-phase antigen. On the third day after the final immunization, splenocytes were isolated and fused with a hypoxanthine–aminopterin–thymidine (HAT)sensitive mouse myeloma cell line (Ag14-sp2/0), by the PEG method. Hybridomas secreting MAbs that were reactive to GA were cloned by the limited dilution method. Established hybridomas were cultured in hypoxanthine–thymidine (HT) medium.
2.2 Instruments An Elx-800 Microplate Reader was used for all absorbance measurements (BioTex Company, NY, USA). An electronic balance (BS124-S) was procured from Sartorius (Gottingen, Germany). Deionized water was prepared using an ultra-class UV plus water purification system (SG Company, Gottingen, Germany). Immunoreactions were carried out in an electrically heated, standing-temperature cultivator (DRP-9082) provided by Samsung Experiment Instrument (Shanghai, China). The refrigerated, high-speed centrifuge was procured C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.5 Determination of isotype of the MAb Isotypes of the MAb have no relation with specificity of antibodies, but are closely related to the purification and application of the antibodies. Determination of the MAb isotypes was carried out according to the instructions of the Mouse Monoclonal Antibody Isotype Kit. Briefly, a sandwich ELISA method was applied to identify the subclasses of monoclonal antibodies against GA. Seven different goat-anti-mouse www.jss-journal.com
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antibodies against isotypes of the antibody (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA) and subtypes of light chain (kappa, lambda) were added into the ELISA plate and incubated at 37⬚C for 1 h. The plate was then washed three times with PBST and blocked with 10% goat serum for 1 h. The culture supernatant of the hybridoma cells was dropped into the plate and incubated at 37⬚C for 1 h. Then the plate was washed with PBST three times, and goat anti-mouse HPR-IgG (1:10000 dilution in PBST) was added into ELISA plate and incubated at 37⬚C for 1 h. The plate was washed with PBST five times, and fresh substrate was then dropped into each well and incubated at 37⬚C for 10 min. The reaction was terminated by adding stopping solution (50 mL, 2 M H2 SO4 ), and the absorbance was measured at 450 nm using a Micro-plate Reader.
2.6 Development of icELISA using anti-GA MAb The reactivity of the MAb against GA-OVA was determined using an indirect ELISA. GA-OVA (1 g/mL, 100 L/well) was adsorbed to the wells of a 96-well immunoplate. In our test, 1% gelatin has good sealing effect to eliminate background noise and has no cross-reactivity with antiserum and MAbs [26, 27]. So the plate was treated with 300 mL of PBS containing 1% gelatin (GPBS) for 1 h to eliminate non-specific adsorption. Then, it was washed three times with PBS containing 0.05% Tween-20 (TPBS). The plate was incubated with 100 L of anti-GA MAb for 1 h followed by 100 L of peroxidase-labeled anti-mouse IgG for 1h. After washing the plate three times with TPBS, 100 L of substrate solution (0.1 M citrate buffer (pH 4) containing 0.015% H2 O2 and 2 mg/mL of 3,3’,5,5’-tetramethyl benzidine (TMB)) was added to each well and incubated for 15 min. Absorbance was measured by a microplate reader at 450 nm. All reactions were carried out at 37⬚C. For indirect competitive ELISA, GA-OVA (1 g/mL, 100 L/well) was adsorbed onto the wells of a 96-well immunoplate and treated with 300 L GPBS for 1 h to reduce non-specific adsorption. 50 L of various concentrations of GA dissolved in 10% methanol were separately incubated with 50 L of MAb for 1 h. The plate was washed three times with TPBS, followed by incubation with 100 L of peroxidase-labeled anti-mouse IgG for 1 h. After washing the plate three times with TPBS, 100 L of substrate solution was added to each well and incubated for 15 min. 50 L/well of stopping solution was then added, and a micro plate reader set to 450 nm was used to measure the resulting absorbance. 2.7 Validation of the icELISA method 2.7.1 Assay sensitivity and assay specificity The icELISA was used with GA-OVA as the solid phased antigen and IgG solution. A range of GA amounts was C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
separately added to compete with the coated antigen. From this competition assay, a standard curve of inhibition for the measured range was created. Furthermore, the cross-reactivities (CRs, %) of GA and structurally related compounds were determined according to Weiler and Zenk’s equation. 
2.7.2 Assay variation Intra-assay variation was tested by evaluating the coefficients of variation (CV, %) for different concentrations of GA plated in six replicates. Inter-assay variation was determined by evaluating GA samples plated in triplicate on three different microtiter plates for three consecutive days.
2.7.3 Recovery Various amounts of GA were added to the blank solution. Therefore, the content of GA in all of the samples was assayed using icELISA, and the recovery rate was obtained.
2.7.4 Correlation between HPLC and ELISA analyses Dried samples (Glycyrrhiza uralensis, 12 g) were pulverized and extracted with 100 mL of 70% ethanol in an ultrasonic bath at 50⬚C for 30 min before filtering. The Agilent 1260 series liquid chromatograph (Agilent Technologies, Palo Alto, CA, USA) is equipped with a quaternary pump, an autosampler and a thermostatically controlled column compartment. The samples were separated using an Agilent ZORBAX SBC18 column (4.6 × 150 mm, 5 m) protected by a C18 guard column (Agilent Technologies). The mobile phase for HPLC analysis consisted of a mixture using water containing 0.2% ammonium acetate buffer (solvent A) and acetonitrile (solvent C) at a constant flow rate of 1.0 mL/min. The gradient profile was: 0–12 min, from 94–78% solvent A; 12–25 min, from 78–75% solvent A; 25–45 min, from 75–60% solvent A. A volume of 10 L of each sample was injected and measured at 260 nm. The sample injection volume was 10 L. A volume of 10 L of each sample was injected and measured at 260 nm. Then, the samples were diluted 100–1000 times and quantified using icELISA. Finally, the interpolated concentrations of the samples obtained by HPLC and ELISA methods were compared.
2.8 Determining GA in Chinese traditional herb prescriptions The GA content of seven iterations of the traditional Chinese prescription was determined. In accordance with the traditional method, the decoctions were extracted with methanol, as described above. We then determined the GA content using proportional dilutions and subsequent ELISA. www.jss-journal.com
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2.9 Knockout of GA by the immunoaffinity column 2.9.1 Preparation of samples and the immunoaffinity column Glycyrrhiza uralensis (3.0 g) was pulverized and extracted with 50 mL of 70% ethanol aqueous solution in an ultrasonic bath at 50⬚C for 15 min and then filtered. The solution was named as GA-extract. According to the GE Healthcare Instructions, the protective additive in the CNBr-activated Sepharose 4B (Pharmacia GE) is removed by washing with 1 mM HCl (pH 4.0, 20 mL/g). The anti-GA MAb was purified by the protein G Fast Flow (FF) method and dialyzed in a 20-fold excess of PBS. The purified anti-GA MAb was coupled to slurry of CNBr-activated Sepharose 4B in coupling buffer (0.1 M NaHCO3 containing 0.5 M NaCl, pH 8.3) and used to prepare the immunoaffinity column. The excess anti-GA MAb removed with five washes with coupling solution followed by five washes with 0.1 M Tris-HCl (pH 8.0 containing 0.5 M NaCl) and 0.1 M acetic acid/sodium acetate (pH 4.0 containing 0.5 M NaCl) buffers for three cycles. 2.9.2 Knockout of GA The extract of Glycyrrhiza uralensis (GA-EXT) was filtered through a 0.45 m PES membrane, and the concentration of GA was determined using HPLC. An appropriate amount of GA-EXT was loaded on the prepared immunoaffinity column and washed five times with washing solvent (0.01 M PBS). The filtrate fractions were collected as the knock-out extract (KO-EXT) and for determination of GA. After the GA signal peak disappeared, elution of GA (E-GA) was initiated with 0.1 M glycine–HCl buffer (containing 0.5 M NaCl, pH 2.7). The collections of E-GA, GA-EXT, and KO-EXT were then filtered through a 0.45m membrane and concentrated. 10 L of the final solutions were analyzed by HPLC.
served as both nebulizing and drying gas (350⬚C, 11 L/min). For full scan MS analysis, the spectra were recorded in the range of m/z 100–1200.
3 Results and discussion 3.1 Synthesis of GA-BSA and GA-OVA conjugates The UV spectra results (Fig. S1) showed that the maximum absorption of GA and BSA samples was located at 253 and 278 nm, respectively. While the peak absorption for GA-BSA was located at 258.1 nm, the shape of the GA-BSA curve exhibited the curve characteristics of both GA and BSA. These results indicated that the GA was successfully conjugated with BSA [29–31].
3.2 Characteristics of MAb against GA A hybridoma secreting an anti-GA MAb named DF5 was produced by fusing splenocytes mouse myeloma cell line (SP2/0-Ag14). As shown in Fig. S2 the MAb DF5 can be identified of IgG1 subtype with kappa light chain.
3.3 Assay sensitivity and specificity An indirect competitive ELISA was established by the MAb DF5. We calculated a standard curve by relating known concentrations of GA to the corresponding absorbance at each concentration. The curve was determined to be linear from 78.12 to 2500 ng/mL (R2 = 0.9973, y = –0.189 ln(x) + 1.7113) (Fig. 1). The value of IC50 was 580 ng/mL, the values of LOD and LOQ were 49.27 and 71.43 ng/mL, respectively, which were calculated by the formula as follows: ALOD =A0 −3∗ SD
ALOQ =A0 −10∗ SD
2.9.3 Determination of E-GA, GA-EXT, and KO-EXT by HPLC and LC–MS HPLC was carried out on an Agilent 1260 series liquid chromatographer (Agilent Technologies, Palo Alto, CA, USA) using an Agilent ZORBAXSB-C18 column (5 m, 0.46 × 150 mm, Agilent, USA). The components of GA-EXT and KO-EXT were separated by gradient elution using water containing 0.2% ammonium acetate buffer (solvent A) and acetonitrile (solvent C) at a constant flow rate of 1.0 mL/min. The gradient profile was: 0–12 min, from 94–78% solvent A; 12–25 min, from 78–75% solvent A; 25–45 min, from 75–60% solvent A. A volume of 10 L of each sample was injected and monitored at 260 nm. LC–MS was carried out on Agilent 1100 LC/MSD Trap XCT Plus System, Agilent ZORBAX SB-C18 column (5 m, 0.46 × 150 mm, Agilent, USA); the mass spectrometer was operated in ESI mode, with the nebulizer and the capillary set at 35 psi, and 4500 V, respectively. High-purity nitrogen C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ALOD is the absorbance of LOD. ALOQ is the absorbance of LOQ. A0 is the absorbance in the absence of the GA. SD is the SD of A0 . Four ELISA of GA have been developed [14, 16, 24] and HPLC  or HPLC–MS [33, 34] are conventionally used for detection GA. Although LOD (49.27 ng/mL) of this method was not the lowest compared with the previous immunoassays of GA, it provided a wide linear range (78–2500 ng) which has many benefits for the GA detection. As shown in Table S4, the newly established ELISA was less sensitive than the LC–MS/MS, but showed strong capacity for measurement without expensive instruments and complex pretreatment. Moreover, ELISA assay required low quantities of samples such as little as 5 L of serum suffices the need for determination in mouse in our previous report  (Table S4). www.jss-journal.com
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Figure 1. Standard curve of inhibition by GA using MAb in icELISA. A is the absorbance in the presence of the NAR and A0 is the absorbance in the absence of the naringin. The data represent the mean of three replicates (mean ± SD; n = 3).
Table 1. Cross-reactivities (%) of anti-GA MAb against naringin and its related compounds
Glycyrrhizic acid Glycyrrhetinic acid Liquiritin Ginsenoside Rg2 Ginsenoside Re Notoginsenoside Saikosaponin D Paeoniflorin Naringin Puerarin Baicalin Hesperidin Cholic acid Deoxycholic acid Chenodeoxycholic acid Astragaloside IV Berberine